Mammalian NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase-5,10-methenyltetrahydrofolate cyclohydrolase is a bifunctional mitochondrial enzyme expressed in most established cell lines but only in developing normal tissues. We report the cloning and molecular characterization of a Drosophila gene (DNMDMC) that encodes a protein with 56% identity to the mammalian bifunctional protein. Like the mammalian bifunctional proteins, the Drosophila protein contains a putative mitochondrial targeting sequence and its transcripts are expressed in developing tissues. Unlike its mammalian homologs, DNMDMC is expressed at high levels in adult tissues. DNMDMC maps to polytene chromosome band 85C, is encoded in three exons, and is closely flanked by two additional genes.