Abstract Porcine calpains (Ca 2+-dependent cysteine proteinases) I and II, which had been purified each to a homogeneous state, were found to hydrolyze specifically carboxyl-terminal amide of substance P and several other biologically active peptidyl amides. This amidaselike activity was demonstrated both by determining released ammonia and by separating products on high-performance liquid chromatography followed by amino acid analysis. The calpain-catalyzed deamidation of substance P occurred exclusively at the carboxylterminal amide, leaving the side-chain glutamine intact. Enkepharinamide and MSH-release inhibiting factor were scarcely deamidated. Calpains I and II showed similar specificities for these amide substances and similar profiles of inhibitions by various protease inhibitors, but distinctly different Ca 2+ requirements. The specificity constants, k cat K m , for substance P were found to be three to four orders of magnitude higher than those for the synthetic substrates.