Many insect pest species have developed insecticide resistance through modifications of acetylcholinesterase. Seven mutations, issued from one nucleotide change, have been associated with resistance in natural populations of Drosophila and housefly (A. Mutero, M. Pralavorio, J. M, Bride, and D. Fournier, Resistance-associated point mutations in insecticide insensitive acetylcholinesterase, Proc. Nail. Acad Sci. USA 91, 5922 (1994); A. L. Devonshire, F. J. Byrne, G. D. Moores, and M. S. Williamson, Biochemical and molecular characterisation of insecticide- insensitive acetylcholinesterases in resistant insects, in "Structure and function of cholinesterases and related proteins- (B. P. Doctor, P. Taylor, D. M. Quinn, R. L. Rotundo, and M, K. Gentry, Eds.), pp. 491-496, Plenum Press, New York, 1998). In order to study the number of mutations which can lead to resistance, we first analyzed the effects of a set of amino acid replacements in the Drosophila acetylcholinesterase on inhibition by several carbamate and organophosphate insecticides. It appeared that most of the mutations led to a reduced sensitivity to insecticides. Second, we investigated the effect of mutations on substrate hydrolysis. We found that most of the variants retained sufficient levels of substrate hydrolysis. These data suggest that more mutations in acetylcholinesterase may be involved in organophosphate and carbamate resistance in addition to the previously known seven mutations.