The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent that one of these, a chymoelastase PR1, is a determinant of pathogenicity. We have shown previously that the wide-domain regulatory circuits of carbon and nitrogen derepression regulate PR1 production. In the present work we have established in addition that PR1 is specifically induced by insect cuticle, but not by other soluble or insoluble proteinaceous substrates. The feeding of elastin or collagen to derepressed established mycelium (starved for carbon and nitrogen) did not enhance PR1 production significantly and the soluble proteins BSA and gelatin rapidly and completely repressed PR1. The carbohydrate polymers cellulose and xylan gave derepressed basal levels of PR1. However, addition of locust cuticle enhanced PR1 production to a level approximately 10-fold that of derepressed mycelium. In order to establish if the enhancing effect of insect cuticle on PR1 production was due to specific induction or merely a reflection of enhanced growth on this insoluble dual carbon and nitrogen source, ergosterol was used as a measure of fungal growth. Expressing enzyme activity per mg dry weight showed that PR1 production in cuticle cultures increased approximately five- and ninefold after 12 and 24 h growth compared with elastin-grown cultures. Thus, the substantial increase in PR1 production on cuticle was shown not to be a function of fungal growth and this confirms that PR1 is induced by a component of insect cuticle; we believe this is the first report of induction by a specific substrate for any microbial protease.