The opportunistic fungus Pneumocystis is the etiologic agent of an interstitial plasma cell pneumonia that primarily afflicts immunocompromised individuals. Like other fungi Pneumocystis maintains a H(+) plasma membrane gradient to drive nutrient uptake and regulates intracellular pH by ATP-dependent proton efflux. Previously, we identified a Pneumocystis gene, PCA1, whose predicted protein product was homologous to fungal proton pumps. In this study, we show by functional complementation in a Saccharomyces strain whose endogenous PMA1 proton pump activity is repressed that the Pneumocystis PCA1 encodes a H(+)-ATPase. The properties of PCA1 characterized in this system closely resemble those of yeast PMA1. Yeast expressing PCA1 grow at low pH and are able to acidify the external media. Maximal enzyme activity (V(max)) and efficiency of substrate utilization (K(m)) in plasma membranes were nearly identical for PCA1 and PMA1. PCA1 contains an inhibitory COOH-terminal domain; removal of the final 40 amino acids significantly increased V(max) and growth at pH 6.5. PCA1 activity was inhibited by proton pump inhibitors omeprazole and lansoprazole, but was unaffected by H(+)/K(+)-ATPase inhibitor SCH28080. Thus, H(+) homeostasis in Pneumocystis is likely regulated as in other fungi. This work also establishes a system for screening PCA1 inhibitors to identify new anti-Pneumocystis agents.