A reversed-phase isocratic high-performance liquid chromatographic assay for the measurement of adriamtcin, adriamycinol and their 7-deoxyaglycones in human serum was described. The lower limit of detection in serum was 3 ng/ml for adriamycin and 1 ng/ml for adriamycinol and the 7-deoxyaglycones with coefficients of variation for k'' of < 5% throughout the day. An extraction technique for serum was described which is capable of an almost equal recovery (> 77%) of adriamycin, metabolites and daunorubicin (the internal standard) without interference from endogenous components of serum. Serum concentrations of metabolites 15 min after i.v. bolus administration of 40 mg/m2 adriamycin in 2 different patients were 26.5 and 16.6 ng/ml for adriamycinol; 109.8 and 5.8 ng/ml for the adriamycinol 7-deoxyaglycone and 21.4 and 17.1 ng/ml for the adriamycin 7-deoxy-aglycone. A total of 6 metabolites of adriamycin were detected in the 2 patients using this methodology.