Abstract A highly purified preparation of the enzyme, β-N-acetylglucosaminidase, has been obtained in a four-step purification procedure from the germinating seeds of Phaseolus vulgaris. The enzyme hydrolyzes p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside. The pH optimum for this reaction is 4.6, and apparent Km and Vmax values are 4.7 x 10-4 m and 7 µmoles per min per mg, respectively. The preparation also shows β-N-acetylgalactosaminidase activity. The enzyme has no action on phenyl 2-acetamido-2-deoxy-α-d-glucopyranoside and, therefore, appears to be specific for glycosidic bonds of β configuration. It cleaves the nonreducing terminal 2-acetamido-2-deoxy-d-glucose residues from glycopeptides derived from desialyzed fetuin and orosomucoid; these glycopeptides had been previously treated with β-galactosidase. The enzyme also releases 1 of the 2 residues of 2-acetamido-2-deoxy-d-glucose from the oligosaccharide chain of the intact bovine pancreatic ribonuclease B.