N-Acetyl-beta-hexosaminidase A was purified 1000-fold from human urine by chromatography on DEAE-Sephadex followed by concanavalin A--Sepharose affinity chromatography. The optimal pH range was 4.4--4.5 for both the N-acetylglucosamine and N-acetylgalactosamine derivatives. The Km values were 0.51 mM and 0.28 mM respectively for the N-acetylglucosamine and N-acetylgalactosamine derivatives. The glycoprotein nature of the urinary enzyme was established by its affinity towards concanavalin A as well as by the presence of sialic acid, galactose, glucose, mannose and hexosamines in the molecule.