We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine--all three at 2.7 A resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 A from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is approximately 50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.