pmaA, an Aspergillus nidulans gene encoding a P-ATPase, has been cloned by heterologous hybridization with the yeast PMA1 gene. The putative 990-residue PmaA polypeptide shows 50% identity to Saccharomyces cerevisiae and Neurospora crassa plasma membrane H(+)-ATPases and weak (19-26%) identity to other yeast P-type cation-translocating ATPases. PmaA contains all catalytic domains characterizing H(+)-ATPases. pmaA transcript levels are not regulated by PacC, the transcription factor mediating pH regulation, and were not significantly affected by an extreme creAd mutation resulting in carbon catabolite derepression. Deletion of pmaA causes lethality, but a single copy of the gene is sufficient to support normal growth rate in pmaA hemizygous diploids, even under acidic growth conditions. As compared to other fungal H(+)-ATPases, PmaA presents three insertions, 39, 7, and 16 residues long, in the conserved central region of the protein. Two of these insertions are predicted to be located in extracellular loops and might be of diagnostic value for the identification of Aspergillus species. Their absence from most mammalian P-type ATPases may have implications for antifungal therapy.