We investigated the pharmacokinetic behavior of carzelesin in 31 patients receiving this drug by 10-min intravenous infusion in a Phase I clinical trial, which was conducted at institutions in Nijmegen (institution 1) and Brussels (institution 2). The dose steps were 24, 48, 96, 130, 150, 170, 210, 250, and 300 μg/m2. Carzelesin is a cyclopropylpyrroloindole prodrug that requires metabolic activation via U-76,073 to U-76,074. The lower limit of quantitation (LLQ) of the high-performance liquid chromatography (HPLC) method used in this study was 1 ng/ml for the parent drug and its metabolic products. Carzelesin was rapidly eliminated from plasma (elimination half-life 23 ± 9 min; mean value ± SD). At all dose levels, U-76,073 was found as early as in the first samples taken after the start of the infusion. However, the concentration of U-76,074 exceeded the LLQ for only short periods and only at the higher dose levels. Although the plasma levels of all three compounds were well above the respective IC50 values obtained by in vitro clonogenic assays, they were much lower than those observed in a preclinical study in mice. There was a substantial discrepancy in the mean plasma clearance␣observed between patients from institution 1 (7.9 ± 2.1 l h−1 m−2) and those from institution 2 (18.4 ± 13.6 l h−1 m−2; P = 0.038), probably reflecting problems with drug administration in the latter institution. The results recorded for patients in institution 1 indicated that the AUC increased proportionately with increasing doses. There was a good correlation between the maximal plasma concentration and the AUC, enabling future monitoring of drug exposure from one timed blood sample. Urinary excretion of carzelesin was below 1% of the delivered dose.