The chitin synthase structural gene WdCHS2 was isolated by screening a subgenomic DNA library of Wangiella dermatitidis by using a 0.6-kb PCR product of the gene as a probe. The nucleotide sequence revealed a 2,784-bp open reading frame, which encoded 928 amino acids, with a 59-bp intron near its 5' end. Derived protein sequences showed highest amino acid identities with those derived from the CiCHS1 gene of Coccidioides immitis and the AnCHSC gene of Aspergillus nidulans. The derived sequence also indicated that WdChs2p is an orthologous enzyme of Chs1p of Saccharomyces cerevisiae, which defines the class I chitin synthases. Disruptions of WdCHS2 produced strains that showed no obvious morphological defects in yeast vegetative growth or in ability to carry out polymorphic transitions from yeast cells to hyphae or to isotropic forms. However, assays showed that membranes of wdchs2 mutants were drastically reduced in chitin synthase activity. Other assays of membranes from a wdchs1wdchs3wdchs4 triple mutant showed that their residual chitin synthase activity was extremely sensitive to trypsin activation and was responsible for the majority of zymogenic activity. Although no loss of virulence was detected when wdchs2 strains were tested in a mouse model of acute infection, wdchs2wdchs3 disruptants were considerably less virulent in the same model, even though wdchs3 strains also had previously shown no loss of virulence. This virulence attenuation in the wdchs2wdchs3 mutants was similarly documented in a limited fashion in more-sensitive cyclophosphamide-induced immunocompromised mice. The importance of WdChs2p and WdChs3p to the virulence of W. dermatitidis was then confirmed by reconstituting virulence in the double mutant by the reintroduction of either WdCHS2 or WdCHS3 into the wdchs2wdchs3 mutant background.