Insects are known to degrade readily most organophosphate insecticides in vivo. In vitro hydrolysis of certain organophosphates was effected by insect homogenates, particularly in the presence of manganic ion as an activator. Purification of house fly esterases showed that the enzymes hydrolyzing the dialkyl phosphoric anhydrides can be at least partially separated from the choline, aliphatic and aromatic esterases, and that more than one house fly esterase acts on the phosphates. The organophosphate substrate specificity is considered for homogenates of several insects and mammalian tissues. These organophosphate-hydrolyzing esterases of insects are discussed with respect to the species-specificity in poisoning by organophosphates and the acquired resistance of house flies to certain organophosphates.