Calpain is a cytosolic cysteine endopeptidase that has been implicated in a number of disorders including cancer. We have synthesized and studied the mu-calpain inhibitory activity and cytotoxicity of peptidyl aldehydes and peptidyl alpha-ketoamides with N-substituted D-proline or L-thiaproline residues at the P2-postion. The most potent and most selective members of the series were (R)-1-(4-nitrophenylsulfonyl)-N-((R,S)-1-oxo-3-phenylpropan-2-yl)pyrrolidine-2-carboxamide (1j) and (R)-1-(4-iodophenylsulfonyl)-N-((R,S)-1-oxo-3-phenylpropan-2-yl)pyrrolidine-2-carboxamide (1n). The compounds inhibited mu-calpain with Ki values of 0.02 microM and 0.03 microM, respectively, and displayed over 180-fold (1j) and 130-fold (1n) greater affinity for mu-calpain compared to cathepsin B. The cytotoxic effect of the compounds was evaluated in two leukemia cell lines (Daudi and Jurkat) and three solid tumor cell lines (DU-145, PC-3, and HeLa). Generally the compounds were modestly cytotoxic and displayed no correlation between the cytotoxic activity and mu-calpain inhibition.