The nature of secreted aminopeptidases inTrichophyton rubrumwas investigated by using a reverse genetic approach.T. rubrumgenomic and cDNA libraries were screened withAspergillusspp. andSaccharomyces cerevisiaeaminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted byAspergillus fumigatususing a recombinant protein fromPichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58–65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 andA. fumigatusorthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities toA. fumigatusorthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced byT. rubrumin a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.