Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, A_1a, A_1b, A_2a, A_2b, A_3a, A_3b, A_4a, and A_4b. Although a base-line resolution of the known four major antimycins A₁, A₂, A₃, and A₄ was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.