β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the culture filtrate of Penicillium oxalicum by fractionation with ammonium sulfate followed by successive column chromatographies with DEAE-cellulose, hydroxylapatite, Sephadex G-150 and Con A-Sepharose 4B. The purified enzyme was found to be homogeneous on polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme showed about 1.5-fold higher β-N-acetylgalactos-aminidase activity than β-N-acetylglucosaminidase activity. The two activities could not be separated by any process and their ratio remained almost constant throughout the whole purification procedure. The molecular weight was determined to be about 143,000 by gel filtration and 141,000 by sedimentation equilibrium. The enzyme was a glycoprotein composed of two subunits having the same molecular weight of about 68,000. The two activities were affected to the same extents by pH and temperature. The optimum pH was 3.0 ~ 4.5 and the stable pH range was 7.0 ~ 9.0, The en...