The in vivo and in vitro metabolism of [14C]malathion was studied in susceptible (LS) and malathion resistant (RM) strains of the sheep blowfly, Lucilia cuprina (Wiedemann). No difference was found between strains in the penetration, excretion, storage, or inhibitory potency of the insecticide. However, RM degraded malathion to its α- and β-monocarboxylic acid metabolites more rapidly than LS, both in vivo and in vitro. This enhanced degradation of [14C]malathion occurred in vitro in both mitochondrial and microsomal fractions of resistant flies. Kinetic analysis revealed that these fractions degraded malathion by discrete mechanisms. The enzymes from the mitochondria of both strains had the same Km, whereas the microsomal enzyme from the RM strain had a fivefold higher Km than that from the LS strain. Studies of esterase activities and the effect of enzyme inhibitors showed that both the mitochondrial and microsomal resistance mechanisms were the result of enhanced carboxylesterase activity. It was concluded that increased carboxylesterase detoxification of malathion adequately explained the high level of malathion resistance in RM if rate-limiting factors such as cuticular penetration were taken into account.