Metarhizium anisopliae infects a broad range of insects by direct penetration of the host cuticle. To explore the molecular basis of this process, its gene expression responses to diverse insect cuticles were surveyed, using cDNA microarrays constructed from an expressed sequence tag (EST) clone collection of 837 genes. During growth in culture containing caterpillar cuticle (Manduca sexta), M. anisopliae upregulated 273 genes, representing a broad spectrum of biological functions, including cuticle-degradation (e.g. proteases), amino acid/peptide transport and transcription regulation. There were also many genes of unknown function. The 287 down-regulated genes were also distinctive, and included a large set of ribosomal protein genes. The response to nutrient deprivation partially overlapped with the response to Man. sexta cuticle, but unique expression patterns in response to cuticles from another caterpillar (Lymantria dispar), a cockroach (Blaberus giganteus) and a beetle (Popilla japonica) indicate that the pathogen can respond in a precise and specialized way to specific conditions. The subtilisins provided an example of a large gene family in which differences in regulation could potentially allow virulence determinants to target different hosts and stages of infection. Comparisons between M. anisopliae and published data on Trichoderma reesei and Saccharomyces cerevisiae identified differences in the regulation of glycolysis-related genes and citric acid cycle/oxidative phosphorylation functions. In particular, M. anisopliae has multiple forms of several catabolic enzymes that are differentially regulated in response to sugar levels. These may increase the flexibility of M. anisopliae as it responds to nutritional changes in its environment.