The effect of incorporating alpha,alpha'-diethylglycine and alpha-aminocyclopentane carboxylic acid at the P(2) position of inhibitors on mu-calpain inhibition was studied. Compound 3 with alpha,alpha'-diethylglycine was over 20-fold more potent than 2 with alpha-aminocyclopentane carboxylic acid. Additionally, 3 was over 35-fold selective for mu-calpain compared to cathepsin B, while 2 was 3-fold selective for cathepsin B compared to mu-calpain. Thus, the conformation induced by the P(2) residue influenced the activities of the compounds versus the closely related cysteine proteases, and suggests an approach to the discovery of selective mu-calpain inhibitors.