Subunit A of the vacuolar proton pump appears to be responsible for the ATP hydrolysis which is coupled to the pumping of protons into a variety of intracellular acid compartments, including the fungal vacuole. We report here the cloning and sequence determination of the gene encoding subunit A from Candida tropicalis. Southern blot hybridization analysis indicates that there is a single gene which encodes this protein. The gene contains a single intron at the extreme 5'-end of the coding region. The gene is predicted to encode a polypeptide of 1088 residues with a calculated molecular mass of 119,019 daltons, yet the mature polypeptide appears to be approximately 67 kDa, indicating that this protein probably undergoes the same sort of processing that is evidenced in the homologous protein from Saccharomyces cerevisiae in which an approximately 50-kDa polypeptide (the spacer) is spliced out of the mature protein. The Candida gene, with and without this middle portion, has been expressed in S. cerevisiae and found to restore a Saccharomyces subunit A deletion mutant (tfp1-delta 8) to apparently wild-type growth at pH 7.6, and normal vacuolar acidification. The peptide sequence of the two predicted mature ends is very similar to the sequences of the analogous proteins from Daucus carota, S. cerevisiae, and Neurospora crassa (60.5, 87.4, and 72.9% identity, respectively), but the middle portion bears only very limited homology with the Saccharomyces protein sequence. Processing of the gene product occurs in S. cerevisiae, Escherichia coli, and in rabbit reticulocyte-mediated in vitro translation, indicating that the excision is probably autocatalytic. The limited sequence identity seen between the Saccharomyces and Candida spacer domains may considerably narrow the functionally important regions responsible for the excision event.