Meperidine carboxylesterase activity was assayed in subcellular fractions of mouse and human liver by coupling the hydrolytic production of ethanol to the reduction of a tetrazolium dye. In mouse liver, the activity was found to be distributed among the mitochondrial, light mitochondrial, and microsomal fractions, whereas in human liver activity was found only in the microsomal fraction. The meperidine carboxylesterases in mouse liver and human liver were inhibited by two irreversible serine hydrolase inactivators (diisopropyl fluorophosphate and paraoxon) and by a reversible transition state analog (trifluoromercaptophenylacetone). Compared to the activities in mouse and human liver microsomes, the activity in mouse liver mitochondria was highly sensitive to the three inhibitors.