Three β-N-acetyl-D-glucosaminidases (E. C. 3. 2. 1. 30) of one B form an d two A forms were isolated from human semen. The enzyme from human seminal plasma was fractionated by (NH₄)₂SO₄. By DEAE-cellulose chromatography, β-N-acetyl-D-glucosaminidase B was eluted with buffer alone and A₁ form and A₂ form of β-N-acetyl-D-glucosaminidase were eluted with 0.1 M NaCl and 0.25 M NaCl respectively. Each enzyme was further purified by Pll cellulose phosphate and Sephadex G-200 chromatography. The highly purified β-N-acetyl-D-glucosaminidase B showed one major protein band and A₁ form and A₂ form showed one major and one minor bands on disc gel electrophoresis at pH 8.3. Three of the enzyme had maximum activities at pH 4.5, but the temperature optimum was 50-54 ℃ for B, 43-47 ℃ for A1 and A2. The three enzymes had identical Km values of 1. 33 mM with p-nitrophenyl-β-N-acetyl-D-glucosaminide as substrate. Sulfite, acetate and β-N-acetyl-D-glucosaminidaseinhibited the β-N-acetyl-D-glucosaminidase B, A₁ and A₂ activities. N-acetyl-n-glucosmine inhibited β-N-acetyl-D-glucosaminidase competitively and the KI values were 1. 64 mM for β-N-acetyl-D-glucosaminidase B and 1. 96 mM for β-N-acetyl-D-glucosaminidase A by Dixon plot. The molecular weight of human semen β-N-acetyl-D-glucosaminidase was 170,000 by gel filtration. The DEAE-cellulose fractions which showed β-N-acetyl-D-glucosaminidase B activity retained 70% of its activity at 60 ℃ for 4 hrs, whereas A₁ and A₂ were heatlabile. However incubation at 60 ℃ for 30 ruin completely inactivated the three purified enzymes.