Calpains are intracellular cysteine proteases with important physiological functions. Up- or downregulation of their expression can be responsible for several diseases, therefore specific calpain inhibitors may be considered as promising candidates for drug discovery. In this paper we describe the synthesis and characterization of a new class of inhibitors derived from the analysis of amino acid preferences in primed and unprimed sites of calpains by incorporation of l- or d-epoxysuccinyl group (Eps). Amino acids for replacement were chosen by considering the substrate preference of calpain 1 and 2 enzymes. The compounds were characterized by RP-HPLC, amino acid analysis and ESI-MS. Selectivity of the compounds was studied by using calpain 1 and 2; and cathepsin B. We have identified five calpain specific inhibitors with different extent of selectivity. Two of these also exhibited isoform selectivity. Compound NH2-Thr-Pro-Leu-(d-Eps)-Thr-Pro-Pro-Pro-Ser-NH2 proved to be a calpain 2 enzyme inhibitor with at least 11.8-fold selectivity, while compound NH2-Thr-Pro-Leu-(l-Eps)-Ser-Pro-Pro-Pro-Ser-NH2 possesses calpain 1 enzyme inhibition with at least 4-fold selectivity. The results of molecular modeling calculations suggest that the orientation of the bound inhibitor in the substrate binding cleft is markedly dependent on the stereochemistry of the epoxysuccinyl group.