A biochemical and histochemical investigation with specific substrates and inhibitors was used to visualize protease, esterase and aminopeptidase activities produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae. Two endoproteases, and aminopeptidase and esterase activities, were mainly localized in simple and complex appressoria and germinating conidia. The effect of inhibitors on two characterized proteases (Pr1 and Pr2) and aminopeptidase activity in appressorial plates was quantified by microdensitometric measurement of reaction products. Pr1 and Pr2 activities were differentially inhibited by various protease inhibitors. Pr1, Pr2, esterase, aminopeptidase and N-acetylglucosaminidase (exochitinase) activities were present during penetration as detected directly following desorption from fungal and cuticle components. The proteases produced in situ were fractionated, and were shown by immunological and enzymological criteria to be the same as those produced in culture media. The sequence of enzyme appearance in situ showed that production of proteolytic enzymes precedes exochitinase production. No production of endochitinase was found before or during hyphal penetration of the cuticle.