Studies of the contribution of enzymatic activity to biological control of plant pathogens have usually relied on enzymes purified from late log phase or stationary phase cultures and ignored the possibility that different enzymes may be produced at different times. We found Two distinct extracellular beta-N-acetylhexsosaminidases in culture fluid of the biocontrol fungus Gliocladium virens The enzymes were purified to apparent homogeneity based on native polyacrylamide gel electrophoresis (PAGE), and purification as great as 150-fold with yields up to 38% were obtained. The beta-N-acetylhexosaminidase purified from 24-h-old cultures (NAH1) migrated as a single band with M(r) = 70K in SDS-PAGE, had pI = 4.5, and lost 50% of its activity at 50 degrees C in 87 min. The beta-N-acetylhexosaminidase from 8-day-old cultures (NAH2) resolved into three bands with M(r) = 75, 68, and 60K, had pI = 5.7, and lost 50% of its activity at 37 degrees C in 82 min. The kinetic parameters for chitobiose were K-m = 2.2 mM, V-max = 0.023 (NAH1) and K-m = 2.8 mM, V-max = 0.015 (NAH2). Among the p-nitrophenyl-containing substrates tested both enzymes showed a preference for p-NP-N-acetyl-beta-D-glucosaminide (K-m = 0.12 and 0.14 mM, respectively). Carbohydrate staining of the purified proteins and changes in electrophoretic mobility after digestion with peptide-N-glycosidase F (PGNase F) or endoglycosidase H (Endo H-f) showed that both enzymes were glycosylated.