Three metabolites of the new antitumor anthracycline epirubicin (4''-epidoxorubicin, 4''-epiDX) detected by HPLC [high-performance liquid chromatography] in urine of 4 patients with mammary carcinoma treated with 75 mg/m2 of the drug, were isolated and identified. In an initial step, fluorescent metabolites present in urine, pooled during the 1st 24 h after drug administration, were adsorbed on a column of a styrene divinylbenzene adsorbent. The subsequent gradient elution with aqueous methanol, followed by extraction with 1-butanol at different pH values, allowed separation of 4''-epiDX and its 13-dihydro derivative from the more polar metabolites. Each anthracycline was subsequently separated by reverse phase liquid chromatography, characterized by acid and enzymatic hydrolyses and studied with chemical-physical analysis. In addition to the parent drug, its 13-dihydro derivative and 4''-O-.beta.-D-glucuronyl-4''-epiDX which were structurally characterized, a glucuronide conjugate of 13-dihydro-4''-epiDX was identified. In 4 patients analyzed, approximately 12% of the total administered dose was estimated, by reverse phase HPLC and fluorescence detection, to be excreted in the 0-24-h pooled urine. 4''-EpiDX accounted for 57% of the total excreted fluorescent anthracyclines, its 4''-O-.beta.-D-glucuronide for 32%, 13-dihydro-4''-epiDX for 7% and its glucuronide for 3%, average values.