The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl-CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila, pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His268, His393, and His426. Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His268Leu-ChAT, His268Gln-ChAT, and His393Gln-ChAT and inactive His393Leu-ChAT, His426Leu-ChAT, and His426Gln-ChAT. The kinetic constants Km(CoA), Km(acetylcholine), and Vmax were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His393Leu-ChAT is disrupted but conserved in His426Leu-ChAT and His426Gln-ChAT. These results suggest that His426 is an essential catalytic residue and could serve as an acid/base catalyst.