4-L-Aspartylglycosylamine amido hydrolase (Amidase) prepared from hog serum and kidney was studied for its substate specificities with synthetic 4-L-aspartylglycosyl-amines in which N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose, and glucose are present as the carbohydrate moieties; and with other aspartyl derivativies including aspartylcyclohexylamine, aspartylaniline, asparthydroxamate, asparthydrazide, and asparagine. Except for aspartylcyclohexylamine and aspartylaniline, which served as competitive inhibitors, the compounds tested were degraded by Amidase at various rates. The enzymes from serum and kidney differed in their relative reaction rates and Michaelis constants for each substrate. Formation of an aspartyl-enzyme as an intermediate was demonstrated by carrying out the enzyme reaction in the presence of hydroxylamine.