Mechanism-based inhibitors of human cytomegalovirus (HCMV) protease have been designed based on the pyrrolidine-5,5-trans-lactam ring system. New routes to the beta-methyl-, desmethyl-, and alpha-methyl-pyrrolidine-5,5-trans-lactam templates have been developed from 2,4-diaminobutyric acid. ESI/MS studies have shown that these inhibitors can bind covalently and reversibly to the viral enzyme in a time-dependent manner by a mechanism which is consistent with acylation of HCMV deltaAla protease at the active site nucleophile Ser 132. SAR in this series of pyrrolidine-5, 5-trans-lactams has defined the relative stereochemisty of the methyl substituent adjacent to the lactam carbonyl, the functionality on the lactam nitrogen, and the mechanism of action of this novel series of serine protease inhibitors against the HCMV deltaAla protease. Activity decreases on moving from the alpha-methyl to the desmethyl to the beta-methyl series. This selectivity is the opposite of that observed for these templates against the elastase and thrombin enzymes. The activity against HCMV deltaAla protease is the greatest with inhibitors based on the Cbz-protected alpha-methyl-5,5-trans-lactam template which have low micromolar activity against the viral enzyme.