A sensitive high-performance liquid chromatography (HPLC) method for the determination of topotecan and total levels of topotecan (lactone plus its ring-opened hydroxycarboxylate form) was developed by the authors and used in several pharmacokinetics studies. During the analysis of plasma and urine samples collected in those studies, an additional peak eluting just after topotecan was observed. Approximately 100 ng of this potential metabolite was isolated from human urine using a solid-phase extraction procedure and purification by HPLC. Analysis of the isolated material by HPLC showed it to be approximately 95% pure. Mass spectrometry data along with the HPLC retention data and fluorescence data (in comparison with synthetic reference standard) are consistent with the metabolite's being N-desmethyl topotecan. The maximal concentrations of metabolite detected in human plasma and urine were relatively low. When topotecan was given as a 30-min infusion at 1.0 mg/m2 daily for 5 days every 3 weeks, the maximal plasma metabolite concentration (lactone plus the ring-opened hydroxycarboxylate form) was about 0.7% (n = 4) of the maximal total topotecan concentration. The average amount of metabolite excreted in urine during the treatment was 1-4% (n = 20) of the delivered dose.