Aspergillus fumigatus is an opportunistic fungal pathogen that infects immunocompromised patients. A putative aspartic protease gene (ctsD; 1425 bp; intron-free) was identiWed and cloned. CtsD is evolutionarily distinct from all previously identiWed A. fumigatus aspartic proteases. Recombinant CtsD was expressed in inclusion bodies in Escherichia coli (0.2mg/g cells) and subjected to extensive proteolysis in the baculovirus expression system. Activation studies performed on puriWed, refolded, recombinant CtsD resulted in protease activation with a pHopt4.0 and speciWc activityD10 U/mg. Pepstatin A also inhibited recombinant CtsD activity by up to 72% thereby conWrming classiWcation as an aspartic protease. Native CtsD was also immunologically identiWed in culture supernatants and puriWed from fungal cultures using pepstatin–agarose aYnity chromatography (7.8 g CtsD/g mycelia). In A. fumigatus, semi-quantitative RTPCR analysis revealed expression of ctsD in minimal and proteinaceous media only. Expression of ctsD was absent under nutrient-rich conditions. Expression of ctsD was also detected, in vivo, in the Galleria mellonella virulence model following A. fumigatus infection. © 2006 Elsevier Inc. All rights reserved.