Resolution of the components of tunicaminyluracil-based antibiotics by reversed-phase high-performance liquid chromatography is described. Two systems are employed using a silica-based ODS bonded-phase support, and gradient elution with either water-methanol or water-methanol-tetrahydrofuran as mobile phase. The ternary mixture reduces analysis time by a factor of 4 whilst retaining resolution, however the increase in background absorption due to introduction of tetrahydrofuran reduces optimum detector sensitivity about sixteen-fold. Both systems are capable of separating the homologues within individual antibiotics and further resolving them into their anteiso-, iso- or normal-isomers as defined by the termination of the fatty acid portion of their structure, The resultant reproducible pattern of peaks in the chromatograms, and the retention time changes associated with catalytic reduction, have allowed assignment of structures to previously unrecognized or unidentified components in all antibiotics studied, e.g., the 23 components identified in MM 19290, and have permitted correlation of the various nomenclatures published for tunicamycin components.