Trypsin is inactivated by the levorotatory enantiomers (most likely PS) of 4-nitrophenyl 4-H-, 4-CH3-,4-OCH3-, and 4-Cl-phenacyl methylphosphonates (PMNs) with second-order rate constants between 231 and 884 M-1 s-1. 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The second-order rate constants for the inactivation of alpha-chymotrypsin by the levorotatory enantiomers of the five PMNs are between 37,000 and 770,000 M-1 s-1, and those for the dextrorotatory enantiomers are between 400 and 640 M-1 s-1; the enantioselectivity is 90-1880. Specific rotation [alpha]22D of the faster-reacting enantiomer of 4-CH3-PMN with trypsin and alpha-chymotrypsin is -30 +/- 6 degrees. 31P NMR of the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the parent compound. Results of molecular mechanics and dynamics calculations show that the principal interactions are between the phosphonyl group and constituents of the oxyanion hole and between the aromatic fragment and residues in the binding regions of the enzymes. Trypsin activity returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the series. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the second to minute time scale. The data support a mechanism of reactivation involving rate-determining intramolecular displacement of Ser by the carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for trypsin reactivation from its adducts indicate involvement of an ionizable group with pKa approximately 8.0. The pH dependence and solvent isotope effects are small in most cases. The compounds demonstrate favorable properties for controllable and temporary modulation of enzyme activity.