Three-dimensional mapping of the interaction of three haloenol lactone enzyme-activated irreversible inhibitors (suicide substrates) with the active site of a-chymotrypsin has been accomplished by using computer graphics combined with molecular mechanics. According to the proposed inactivation mechanism for these lactones, enzyme inhibition is brought about by a two-step process, i.e., transfer of the lactone acyl group to Ser-195 and subsequent alkylation of His-57 by the resulting halomethyl ketone. In order to study this process, a 115 non-hydrogen atom model of the active site of chymotrypsin was constructed with coordinates taken from an X-ray crystallographic structure, and systematic conformational analysis and energy minimization of the mechanism-based haloenol inhibitors in this model active site were performed at three stages: (1) the non-convalent substrate complex (“Michaelis complex”), (2) the acyl enzyme alkylation complex, and (3) the suicide compound (bis-adduct). The results of these studies support the postulated mechanism, and the calculated energies for the three lactones substrates are in good agreement with the availabile inactivation kinetic data. Estimates of van der Waals interaction energy correlate with the inactivation binding constants (Ki), and estimates of the enthalpy of the alkylation reaction parallel the inactivation rate constants (K2). In addition, the calculations are also consistent with other known effects of configurational changes and halogen substitution. Such calculations of the interaction between a potential mechanism-based inhibitor and the active site of the target enzyme may be useful in formulating a plausible mechanism of action and in guiding in the synthesis of new, more potent inhibitors.