A number of maleyl peptide p-nitrobenzyl esters have been synthesized to study elastase-catalyzed hydrolysis reactions. These new substrates were used as acyl donors to investigate the S'-subsite specificity of porcine pancreatic elastase by partitioning of the acyl enzyme between various added nucleophiles and water. The following results were obtained. 1. Porcine pancreatic elastase prefers amino acid residues with small side chains in the P'1 position. 2. The nucleophile binding is improved by a positively charged P'1 side chain, whereas a negatively charged function results in a very low binding tendency. 3. Elongation of the nucleophile to the P'2 position leads to higher aminolysis rates. 4. S' specificity is substantially influenced by the P1 residue of the acyl enzyme.