Pneumocystis Pneumonia is an opportunistic fungal infection that causes severe respiratory impairment in immunocompromised patients. The viability of Pneumocystis organisms is dependent on the cyst cell wall, a structural feature that is regulated by essential cell wall associated enzymes. The formation of the glucan-rich cystic wall has been previously characterized, but glucan degradation and degradation during trophic excystment is not yet fully understood. In this study we have identified a Pneumocystis -1,3-endoglucanase gene (PCEng2) that is demonstrated to play a significant role in cell wall regulation. The full sequence of the gene revealed a 2.2kb open reading frame with conserved amino acid domains homologous to similar fungal glycosyl hydrolases (GH family 81). The gene transcript showed upregulation in cystic isolates and the expressed protein was detected within both cyst and trophic forms. Complementation assays in Eng2 deleted S. cerevisiae strains showed restoration of the cell wall separation defect during proliferation, demonstrating the importance of PCEng2p during fungal growth. These findings suggest that regulation of cyst cell wall -glucans is a fundamental process during completion of the Pneumocystis carinii life cycle.