O6-Benzylguanine and its metabolite, 8-oxo-O6-benzylguanine, are equally potent inhibitors of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase. Pharmacokinetic values are derived from cancer patients participating in a phase I trial (10 or 20 mg/m2 of O6-benzylguanine in a single bolus dose or 10 to 120 mg/m2 as a 60-min constant infusion). A two-compartment model fits the plasma concentration versus time profile of O6-benzylguanine. O6-Benzylguanine is eliminated rapidly from the plasma compartment in humans (t1/2 alpha and t1/2 beta are 2 +/- 2 min and 26 +/- 15 min [mean +/- SD, n = 7], respectively), and its plasma clearance (513 +/- 148 mL/min/m2) is not dose dependent. Metabolite kinetics are evaluated using both a novel approach describing the relationship between O6-benzylguanine and 8-oxo-O6-benzylguanine and classical metabolite kinetics methods. With increasing doses of O6-benzylguanine, the plasma clearance of 8-oxo-O6-benzylguanine, decreases, prolonging elimination of the metabolite. This effect is not altered by coadministration of BCNU. The urinary excretion of drug and metabolites is minimal.