Eleven metabolites of the anthracycline antineoplastic antibiotic aclacinomycin A (Acm) were isolated and purified from the urine of four patients treated with 100-120 mg/m2 of Acm. In an initial step, pooled urine was extracted and Acm and its metabolites were concentrated in n-butanol with a horizontal flow-through coil planet centrifuge. Individual metabolites were subsequently isolated and purified by thin-layer chromatography (TLC). Purified urinary species were characterized and compared to standards of Acm and its known metabolites by TLC, high performance liquid chromatography (HPLC), acid and enzymatic hydrolysis, and mass spectrometry. Nine of the urinary species were identified as either Acm or metabolites previously described in fermentation broths of Streptomyces galileus, in vitro reactions, animal plasma, or human plasma. Another urinary species corresponded to the major and previously unidentified Acm metabolite observed in human plasma. Sufficient quantitites of this material were isolated to allow its characterization and identification as bisanhydroaklavinic acid. The most polar urinary species isolated was identified as a beta-glucuronide conjugate of bisanhydroaklavinic acid. As such, these studies demonstrate the ability of humans to produce all of the previously proposed metabolites of Acm and establish the identity of the major Acm metabolite observed in human plasma.