We measured the plasma concentrations of 7-ethyl-10-[4-(1-piperidino)-1- piperidine]carbonyloxycamptothecin (CPT-11) and the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), after treatment with CPT-11 to rats pretreated with bis-p-nitrophenylphosphate (BNPP) which is a specific inhibitor of carboxylesterase, and non-pretreated rats. The plasma level of SN-38 was decreased in the BNPP-pretreated group compared with these of non-pretreated group, indicating that the esterase involved in CPT-11 metabolism is a carboxylesterase. We also characterized the molecular species of carboxylesterase involved in CPT-11 metabolism using enzyme preparations purified from liver microsomes. Thirteen carboxylesterase isozyme activities towards CPT-11 were compared and guinea pig GLP1 was found to have the highest activity, while human HU1 isozyme had relatively lower activity than those of animal species. In studies on the kinetic parameters of the hydrolysis of CPT-11 by the purified carboxylesterase isozymes the highest Vmax value of the isozymes was found in human HU1 and the smallest was seen in rat RL1. The Vmax/Km for RL1 showed the largest value of 21.7 nmol/mg protein/mM.