Microsporum canis is the main agent of dermatophytosis in dogs and cats and is responsible for frequent zoonosis. The pathogenesis of the disease remains largely unknown, however. Among potential fungal virulence factors are secreted keratinolytic proteases, whose molecular characterization would be an important step towards the understanding of dermatophytic infection pathogenesis. M. canis secretes a 31.5 kDa keratinolytic subtilisin-like protease as the major component in a culture medium containing cat keratin as the sole nitrogen source. Using a probe corresponding to a gene's internal fragment, which was obtained by polymerase chain reaction, the entire gene encoding this protease named SUB3 was cloned from a M. canislambdaEMBL3 genomic library. Two closely related genes, termed SUB1 and SUB2, were also cloned from the library using as a probe the gene coding for Aspergillus fumigatus 33 kDa alkaline protease (ALP). Deduced amino acid sequence analysis revealed that SUB1, SUB2, and SUB3 are secreted proteases and show large regions of identity between themselves and with subtilisin-like proteases of other filamentous fungi. Interest ingly, mRNA of SUB1, SUB2, and SUB3 were detected by reverse transcriptase nested-polymerase chain reaction from hair of experimentally infected guinea pigs. These results show that SUB1, SUB2, and SUB3 encode a family of subtilisin-like proteases and strongly suggest that these proteases are produced by M. canis during the invasion of keratinized structures. This is the first report describing the isolation of a gene family encoding potential virulence-related factors in dermatophytes.