Two full-length cDNAs were isolated from Coccidioides posadasii that encode two deduced proteins (CpHEX1 and CpHEX2) with homology to the glycosyl hydrolase 20 family of beta-N-acetylhexosaminidases. CpHEX1 consists of 595 amino acids, has a predicted molecular mass of 68 kDa and shares the highest identity with the N-acetylhexosaminidase (NAGA) of Aspergillus nidulans, while CpHEX2 consists of 603 amino acids, has a predicted molecular mass of 68.5 kDa and shares the highest identity with NAG1 from Paracoccidioides brasiliensis. CpHEX1 and CpHEX2 share only 23% identity and have dissimilar homologies showing more identity with other fungal beta-N-acetylhexosaminidases than with each other. Phylogenetic analysis of selected beta-N-acetylhexosaminidases placed CpHEX1 in a cluster with the orthologs from A. nidulans, Aspergillus oryzae, Penicillium chrysogenum and Candida albicans, while CpHEX2 grouped with the orthologs from P. brasiliensis and the Trichoderma spp. beta-N-acetylhexosaminidase activity and transcripts encoding CpHEX1 and CpHEX2 were detected in vitro during the spherule-endospore (SE) phase. Expression of the Cphex1 transcript exhibited a temporal increase that correlated with beta-N-acetylhexosaminidase activity, while the Cphex2 transcript remained relatively constant. The addition of N-acetylglucosamine to the cultures increased beta-N-acetylhexosaminidase activity and the expression of the Cphex1 transcript. A native beta-N-acetylhexosaminidase enzyme was purified from in vitro SE phase and identified as CpHEX1 by mass spectrometric analysis. Both the CpHEX1 and CpHEX2 cDNAs were expressed as recombinant fusion proteins and purified under denaturing conditions to apparent homogeneity but they lacked enzymatic activity.