N-(N-acetyl-1-phenylalanyl)aminoacetronitrile is an inhibitor of papain. With 13C NMR spectroscopy we have shown that a reversible covalent adduct is formed with papain. The reversible nature of the covalent-adduct formation was demonstrated with NMR saturation-transfer technique using a DANTE pulse for selective excitation. In addition the covalent adduct was displaced with an aldehyde inhibitor to regenerate the nitrile compound. No hydrolysis of the nitrile was observed. The covalent adduct is most likely a thioimidate formed between the essential thiol and the nitrile. Several p-nitroanilide substrates and their corresponding nitrile inhibitors were examined. A correlation between Ki and kcat/Km was observed. This finding together with the fact that the pH dependence of Ki parallels that of kcat/Km suggests that the interaction of nitriles and papain has considerable transition-state character. In contrast, a nitrile was shown to be an ineffective inhibitor of alpha-chymotrypsin.