Beauveria bassianagrown in a liquid medium containingN-acetyl-D-glucosamine and colloidal chitin produced two distinctN-acetyl-D-glucosaminidases, NAGase 1 and NAGase 2. NAGase 1 had a molecular weight of 97 000 and NAGase 2 was comprised of two subunits, of molecular weights 64 000 and 66 000. The optimal temperature and pH for NAGase 1 were 57 °C and pH 5 and for NAGase 2 they were 37 °C and pH 5. NAGase 1 was more thermostable than NAGase 2. The isoelectric points of NAGase 1 and 2 were ca. pH 9.5 and 5.5, respectively. NAGase 1 and 2 were unaffected by a 10 mM concentration of chloride salts of the ions Ca2+, Mg2+, or Zn2+, by 10 mM EDTA, and by 0.25–1 mM of short chain fatty acids. Dithiothreitol caused some inactivation of NAGase 2 while stimulating activity of NAGase 1. NAGase 1 had aKmof 0.38 mM and aKcat/Kmof 3923.88 M−1∙s−1. NAGase 2 had aKmof 2.095 mM and aKcat/Kmof 411.88 M−1∙s−1whenp-nitrophenyl-β-N-acetylglucosaminide was used as the substrate.Key words:Beauveria bassiana,N-acetyl-D-glucosaminidase, entomopathogenic fungus, extracellular enz