The susceptibility of the American cockroach, Periplaneta americana L. (Blattodea: Blattidae), against malathion was tested in the laboratory. The levels of insecticide detoxifying enzymes among different populations of P. americana were also compared. Standard methods were used for the estimation of non-specific esterases, glutathione S-transferases (GSTs) and monooxygenases. All populations showed resistance against malathion and significantly higher activities of insecticide detoxifying enzymes were recorded as compared to the susceptible population (control). This higher enzymatic activity and elevated levels of GSTs and monooxygenases is the possible reason for malathion resistance in P. americana. However, non-specific esterases and monooxygenases might be involved in the development of resistance in other populations at the same localities. The development of the resistance mechanism was noticed in all tested populations of P. americana and it is an indication to replace malathion insecticides against American cockroaches in the investigated regions especially, and also a warning for other regions in the world.