The staurosporine derivative, N-benzoylstaurosporine (CGP 41 251; I), is a protein kinase C inhibitor that has been selected for phase I clinical evaluation in cancer patients. We have developed a selective and sensitive assay of the drug and three potential metabolites in human plasma. The method is based on reversed-phase high-performance liquid chromatography with fluorescence detection. The sample pretreatment involves liquid-liquid extraction with diisopropyl ether with recoveries over 88%. The limit of detection and limit of quantitation of the parent compound and two metabolites were 0.5 and 1.0 ng/ml, respectively. For the third metabolite the limit of detection and limit of quantitation were 1.0 and 2.0 ng/ml, respectively. Linear calibration lines were obtained over the range of 1-1000 ng/ml. The between-day and within-day precisions were < 7.1% for all the analytes. In plasma the compounds were stable for at least one month if stored at -30 degrees C or below. The applicability of the method for in vivo studies has been demonstrated in a pharmacokinetic study in rat receiving 0.5 mg/kg of the drug as an intravenous bolus injection. Compound I and two metabolites were detected.