Peptidyl alpha-keto amides have been synthesized and tested as inhibitors of the cysteine protease calpain. A stereospecific synthesis was devised in which Cbz-dipeptidyl-alpha-hydroxy amides were oxidized with TEMPO/hypochlorite to the corresponding alpha-keto amides. This oxidation was accomplished in good yields and without epimerization of the chiral center adjacent to the ketone. The potent inhibition of porcine calpain I by the L,L diastereomers, combined with the poor inhibition by the L,D diastereomers, established the requirement for the all-L stereochemistry of the active inhibitor. The early lead inhibitors were very hydrophobic and, therefore, poorly soluble in aqueous solutions. Using the stereospecific route, new compounds were prepared with polar groups at the C- and N-termini. These modifications resulted in more soluble inhibitors that were still potent inhibitors of calpain. Studies of the stability of these alpha-keto amides showed that absolute stereochemistry can be maintained in acidic and unbuffered environments but general base-catalyzed epimerization of the chiral center adjacent to the ketone occurred rapidly. The alpha-hydroxy precursors were inactive as inhibitors of calpain, which supports the hypothesis that the alpha-keto compounds reversibly form an enzyme-bound tetrahedral species that results from the nucleophilic addition of the catalytic thiol of calpain to the electrophilic ketone of the inhibitor.