A series of dipeptidyl and tripeptidyl alpha-keto esters, alpha-keto amides, and alpha-keto acids having leucine in the P2 position were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, cathepsin B, and papain. In general, peptidyl alpha-keto acids were more inhibitory toward calpain I and II than alpha-keto amides, which in turn were more effective than alpha-keto esters. In the series Z-Leu-AA-COOEt, the inhibitory potency decreased in the order: Met (lowest KI) > Nva > Phe > 4-Cl-Phe > Abu > Nle (highest KI) with calpain I, while almost the reverse order was observed for calpain II. Extending the dipeptide alpha-keto ester to a tripeptide alpha-keto ester yielded significant enhancement in the inhibitory potency toward cathepsin B, but smaller changes toward the calpains. Changing the ester group in the alpha-keto esters did not substantially decrease KI values for calpain I and calpain II. N-Monosubstituted alpha-keto amides were better inhibitors than the corresponding alpha-keto esters. alpha-Keto amides with hydrophobic alkyl groups or alkyl groups with an attached phenyl group had the lower KI values. N,N-Disubstituted alpha-keto amides were much less potent inhibitors than the corresponding N-monosubstituted peptide alpha-keto amides. The peptide alpha-keto acid Z-Leu-Phe-COOH was the best inhibitor for calpain I (KI = 0.0085 microM) and calpain II (KI = 0.0057 microM) discovered in this study. It is likely that the inhibitors are transition-state analogs and form tetrahedral adducts with the active site cysteine of cysteine proteases and form hydrogen bonds with the active site histidine and possibly another hydrogen bond donor in the case of monosubstituted amides. Several inhibitors prevented spectrin degradation in a platelet membrane permeability assay and may be useful for the treatment of diseases which involve neurodegeneration.