Cancer chemotherapy is a concern of many people today: laboratory scientists, clinicians, cancer victims and news reporters. Their hopes for a cancer cure are always raised when a new chemotherapeutic agent is discovered in the laboratory and shows some promising characteristics towards clinical application. Some of the agents that have been discovered are of natural origin(1), and are complex structures of unknown composition; others are enzymes or antibiotics. The antibiotics are obtained mostly from fermentations, but are also derived from plants or marine animals. Considerable efforts are being made in laboratories to purify these antibiotics to homogeneity and to determine their structure, mode of action, toxicity and applicability to clinical cancer chemotherapy. All these complex functions require analytical support. One of the most modern analytical tools, high pressure liquid chromatography (HPLC), is used with increasing frequency to analyze these antitumor antibiotics in different media and for different reasons. Before the advent of HPLC, analytical methods used in connection with different antibiotic assays relied on liquid chromatography, countercurrent distribution(2), thin layer chromatography(3) and gas chromatography(4). Liquid chromatography, in general, is a time-consuming, slow technique. Thin layer chromatography is much faster and uses inexpensive instrumentation but lacks resolution and reproducibility for complicated, large compounds and especially for mixtures. Gas chromatography is a fast chromatographic system, with good reproducibility, precision and specificity at relatively modest instrumentation costs. However, many antibiotics cannot be analyzed by gas chromatographic techniques because they cannot be volatilized. In the last decade or so, HPLC has been developed into an analytical tool that combines the advantages of all previous chromatographic techniques. Most of the details of the techniques of HPLC are well described in recent literature(5)(6), and there is no need to deal with them here. Briefly, HPLC uses a narrow column with small diameter column packing particles, high pressure to obtain the flow rate necessary for short analytical time and highly sensitive detectors. The short analytical time is an important factor in analyzing antibiotics, because some antibiotics decompose during lengthy analytical manipulations. Also, analytical results may be needed urgently, e.g., for assessment of antibiotic composition in fermentation broth or in human or animal tissues. This short analytical time is achieved by high volume of solvent flow, which in turn is achieved by high column-inlet pressure. This analytical arrangement is very seldom a problem in connection with antibiotic analysis. The separation of antibiotics is monitored with ultraviolet absorption detectors in most cases, since many of the antibiotics absorb in the ultraviolet range. However, some antibiotics, e.g., the aminoglycosides, have no characteristic ultraviolet absorption above 210 nm. In such cases pre- or postcolumn derivatization is performed to provide antibiotic derivatives which can be monitored by fluorescence or ultraviolet detectors. HPLC has been used increasingly in the antibiotic field in research, quality control and manufacturing environments. For many antibiotics the official or most accepted assay is now performed by HPLC. However, some of the HPLC assays lack precise characterization of the chromatographic system, mostly because the system was applied to non-routine testing. For example, no precise control of the temperature is mentioned in most literature although work done with the vinca alkaloids demonstrated the importance of this parameter(7). Precise characterization of the columns used in connection with antibiotic work is almost entirely lacking. Today HPLC is used in the isolation studies of new antibiotics, in preparation of large quantities of antitumor antibiotics for biological studies and, if the assay proves to be sensitive enough, for quantitative estimation of antibiotics in biological fluids and in various drug preparations. Because some of the work was done with equipment not used any more, these systems need to be translated to more current conditions. Routine assays should undergo collaborative studies before wide-scale introduction, and such studies were done for several antibiotic assays as discussed below. This review summarizes and comments on the HPLC systems used in connection with antitumor antibiotic analysis. It is intended to alert the reader about possible use of HPLC in connection with studying antibiotics rather than as a critical evaluation of work done in other laboratories. Discussions are grouped according to the most important or most frequently analyzed antibiotics and the use of HPLC in isolation studies of new antitumor antibiotics. Moreover, the literature on determination of these compounds in biological fluids has been selected so that similar analytical details are not repeated too frequently. Also, methods older than 8 to 10 years are not reported in this discussion. It will be apparent to the reader that most of the HPLC processes discussed below use reversed phase systems. The most frequently used column is μBondapak C18. UltraSphere ODS, Nucleosil c-18 and Durapak. Occasionally the application of a guard column like Co:Pell ODC or RSiL had to be employed. For eluting solvents combinations of methanol and acetonitrile with phosphate buffers are used most frequently. It should be mentioned that in this review the HPLC nomenclature used is that of the original authors. It was felt that an attempt to modify their descriptions for the purpose of uniformity would create a problem for those who want to refer to the original paper. Also, no attempt was made to describe the origin and quality of reagents used in the experiments. It was assumed that all solvents were filtered and degassed before use. Structures of the discussed antibiotics can be found in the Merck Index, Handbook of Antibiotic Compounds (J. Berdy, A. Aszalos, M. Bostian and K. L. McNitt eds., CRC Press) and in the original articles cited, and have not been duplicated in this paper. Many papers describe the use of HPLC in conjunction with the objective of that paper, e.g., isolation of an antibiotic, metabolic studies, etc. In each case only the HPLC portion of the paper and the objective of the study have been described; details and results of the study were not reviewed here.