Abstract A soluble enzyme with amidohydrolase activity was isolated from the roots of soybean seedlings and purified 45-fold by ammonium sulfate fractionation, gel filtration and cellulose ion-exchange chromatography. The enzyme hydrolyzed primarily N α -benzoyl- dl -arginine p -nitroanilide and to a slight extent the p -nitroanilides of glycine and l -leucine, but it did not hydrolyze casein, Azocoll and the p -nitroanilides of l -lysine, l -cystine, succinyl- l -phenylalanine and glutaryl- l -phenylalanine. The apparnet K m for N α -benzoyl- dl -arginine p -nitroanilide was 5·78 × 10 −5 M. The effects of temperature, pH, metal ions and sulfhydryl reagents on the enzyme were also investigated.