Porcine kidney β-acetylhexosaminidase A was purified to electrophoretic homogeneity by procedures involving ammonium sulfate precipitation, gel filtration and column chromatography on diethylaminoethyl cellulose, octyl Sepharose CL-4B and ε-aminohexanoyl β-acetylglucosaminylamine-Sepharose 4B. Analysis by sodium dodecyl sulfate-gel electrophoresis suggested that the enzyme consists of three subunits with molecular weights of 61000, 31000 and 9000. The molecular weight of the intact enzyme as determined by gel filtration was 129000 at pH 8.0 and 302000 at pH 5.0. Kinetic analysis with mixed substrates as well as inhibition studies with various substrate analogs, indicated that the enzyme hydrolyzes both β-N-acetylglucosaminide and β-N-acetylgalactosaminide at the same active site. Among hexoses tested for inhibitory effect, only mannose was an effective competitive inhibitor. A comparison of mannosides with N-acetylglucosaminides with respect to binding ability to the enzyme in terms of K1 or Km values implied the presence of a site for mannose other than the active site. On the other hand, kinetic studies with mixed inhibitors indicated that mannose competes with N-acetylgalactosamine, N-acetylglucosamine and acetamide.