The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.