The transvacuolar pH gradient determines, to a significant extent, the distribution of the antimalarial drug chloroquine in Plasmodium falciparum. A proton pump, similar to the vacuolar ATPase found in many cell types, appears to regulate a pH gradient across the membranes of acidic compartments of the parasite. In order to understand and define the components involved in the maintenance of the vacuolar pH gradient, we have cloned and characterized a gene, designated VAP B, encoding a P. falciparum homologue of the B subunit of the vacuolar ATPase. The VAP B gene encodes a protein of 494 amino acids which has between 69% and 74% amino acid identity with the sequences of vacuolar ATPase B subunits of other organisms. The VAP B gene exists as a single copy gene on chromosome 4 that gives rise to a RNA transcript of 2.4 kb. Antibodies raised to the VAP B protein react specifically with a protein of 56-kDa, consistent with the size predicted from the gene sequence and with the homologous protein from other organisms. The 56-kDa protein is expressed throughout the asexual life cycle and subcellular localization by indirect immunofluorescence shows that the protein has a heterogeneous distribution over most of the parasite. This suggests that the function of the vacuolar proton ATPase is not confined to the regulation of the pH of the digestive vacuole.