Two forms of N-acetyl-beta-D-glucosaminidase were purified from bovine mammary gland by DEAE-cellulose chromatography, Sephadex G-200 gel filtration, affinity chromatography on Con A-Sepharose and preparative isoelectric focusing. The two forms, designated A and B on the basis of their binding to DEAE-cellulose at pH 7, were glycoproteins with different molecular weights as determined by gel filtration and sedimentation equilibrium analysis. The A form had a molecular weight of 118 000, while the B form had a molecular weight of 234 000. Both A and B forms of the purified enzyme showed the presence of two distinct subunits, having apparent molecular weights of 55 000 and 25 000 as determined by sodium dodecyl sulphate-electrophoresis. Amino acid composition of the purified forms showed that a high degree of similarity existed between the two forms. However, the B form had slightly higher levels of serine and threonine than the A form. The structure and possible interrelationship of these two forms in the bovine mammary gland are discussed in relation to the structure of N-acetyl-beta-D-glucosaminidase from other sources.