A comprehensive ab initio analysis of the gas-phase mechanisms of alkaline hydrolysis for a number of phosphotriesterase substrates-O,O-diisopropyl phosphorofluoridate (DFP), O-isopropyl methyl phosphonofluoridate, O,O-diethyl p-nitrophenyl phosphate (paraoxon), O,O-diethyl p-nitrophenyl thiophosphate (parathion), N-acetyl phosphoramidothioate (acephate), O,O-diethyl S-2-ethylthioethyl phosphorothioate (demeton-S) and O-ethyl N,N-dimethyl phosphoramidocyanidate-has been presented herein. The results indicate that, although an associative mechanism of alkaline hydrolysis is followed by all these compounds, P-F and P-CN bonds are cleaved according to the multistep addition-elimination scheme, whereas the breakage of P-O and P-S bonds appears to be consistent with the one-step direct-displacement mechanism. Of the two alternative reaction pathways present in all those cases (except of acephate), the most probable one involves the proton from a nucleophilic hydroxide experiencing an additional stabilization by the phosphoryl oxygen atom.