Extracellular chitinases have been suggested to be virulence factors in fungal entomopathogenicity. We employed isoelectric focusing and a set of three fluorescent substrates to investigate the numbers and types of chitinolytic enzymes produced by the entomopathogenic fungi Metarhizium anisopliae, Metarhizium flavoviride, and Beauveria bassiana. Each species produced a variety of N-acetyl-(beta)-d-glucosaminidases and endochitinases during growth in media containing insect cuticle. M. flavoviride also produced 1,4-(beta)-chitobiosidases. The endochitinases could be divided according to whether they had basic or acidic isoelectric points. In contrast to those of the other two species, the predominant endochitinases of M. anisopliae were acidic, with isoelectric points of about 4.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the acidic chitinases of M. anisopliae into two major bands (43.5 and 45 kDa) with identical N-terminal sequences (AGGYVNAVYFY TNGLYLSNYQPA) similar to an endochitinase from the mycoparasite Trichoderma harzianum. Use of polyclonal antibodies to the 45-kDa isoform and ultrastructural immunocytochemistry enabled us to visualize chitinase production during penetration of the host (Manduca sexta) cuticle. Chitinase was produced at very low levels by infection structures on the cuticle surface and during the initial penetration of the cuticle, but much greater levels of chitinase accumulated in zones of proteolytic degradation, which suggests that the release of the chitinase is dependent on the accessibility of its substrate.