The cytostatic agent Elsamitrucin is a new fermentation product active in a variety of in vivo tumor models of murine and human origin. To determine its pharmacokinetics during the clinical phase I trial, an HPLC procedure was developed and validated. Plasma samples were extracted after addition of the internal standard, i.e. the analog Chartreusin. Urine samples were injected without extraction of the samples. Because of the wide concentration range of Elsamitrucin in the plasma samples two standard curves were used: up to 100 nM and from 100-1000 nM. Recoveries of Elsamitrucin from plasma were 87% and 74% for concentrations lower and higher than 100 nM, respectively. The detection limits were 1 nM in plasma and 7.5 nM in urine at a signal-to-noise ratio of 3. The accuracy ranged from 95-107% for plasma and from 96-104% for urine. The within-day precision was < or = 4.8% and < or = 2.8% in plasma and urine, respectively. The between-day precision was < or = 4.4% and < or = 7.1% in plasma and urine, respectively. The method proved to be sufficiently sensitive, specific and accurate for analysis of clinical samples for pharmacokinetic purposes.