Adenine nucleotide translocases (ANT) are required for the exchange of ADP and ATP across the inner mitochondrial membrane. They are essential for life, and most eukaryotes have at least two different Ant genes. Only one gene had been described from Drosophila, and this had not been characterized genetically. We show that mutations in this gene correspond to the previously described loci, sesB and l(1)9Ed. Immediately adjacent to this gene is another encoding a second ANT protein, which has 78% identity to that encoded by sesB/l(1)9Ed. These two genes are transcribed from a common promoter, and their mRNAs are produced by differential splicing. Hutter and Karch suggested that the sesB ANT gene corresponded to Hmr, a gene identified by an allele that rescues otherwise inviable interspecific hybrids between Drosophila melanogaster and its sibling species. This hypothesis is not supported by our study of the ANT genes of D. melanogaster.