Prior phytochemical investigations of Catharanthus pusillus (Indian origin) isolated ajmalicine, vindorosine, cardiotoxic pusiline and pusiliniue, three unidentified sterols, and N-benzoyl-β-phenylalaninol. In a continuing study to discover new antitumor agents, the authors analyzed the alkaloids of C. pusillus leaves using modified procedures. The total crude alkaloids (0.6% yield) were divided into six fractions, three of which showed cytotoxicity against Eagle's 9 KB carcinoma of the nasopharynx in cell culture. Column chromatography of the weakly basic Fraction I yielded lochnerinine (as the hydriodide salt), an α-methyleneindoline alkaloid previously reported only from C. roseus and C. lanceus. This compound was found to be responsible for at least part of Fraction I's cytotoxic activity, representing the first isolation of lochnerinine from C. pusillus. Gradient pH fractionation of the nonphenolic tertiary alkaloid Fraction III resulted in the isolation of vindorosine (0.2% of total crude alkaloids, previously exclusive to C. roseus) and ajmalicine (0.04% of total crude alkaloids, present in multiple Apocynaceae species). Both vindorosine and ajmalicine were non-cytotoxic, and the active component in Fraction III remains unidentified. No attempts were made to separate alkaloids from Fractions II, IV, or V.