A Drosophila melanogaster (Dm) cDNA (vha14) encoding the 14-kDa F-subunit of the vacuolar H(+)-ATPase (V-ATPase) has been cloned via homology with the corresponding Manduca sexta (Ms) gene. Its deduced translation product is a 124-amino-acid polypeptide sharing 90% identity with the Ms polypeptide and 50% identity with an analogous polypeptide of Saccharomyces cerevisiae, and a more distant similarity to a subunit of the Na(+)-transporting ATPase of Enterococcus hirae. Homology was also found with expressed sequence tags from man, Arabidopsis thaliana, Caenorhabditis elegans and C. briggsiae, Oryza sativa and Plasmodium falciparum, indicating that the subunit is phylogenetically conserved. The Dm gene (vha14) is present as a single copy at cytological position 52B on the second chromosome, and gives rise to an mRNA species of 0.65 kb. Expression of the latter shows relatively little variation during development, or between adult head, thorax and abdomen, suggesting that the F-subunit is a relatively ubiquitous component of the V-ATPase.