We have carried out experiments to determine the structural organization of dhod and its apparent dihydroorotate dehydrogenase (DHOdehase) product. Germline transformation with dhod genomic DNA sequences permitted assignment of the functional limits of the gene to a 5-kb region, providing an experimental system for detailed analysis of this gene, as well as the DHO dehase protein. As expressed in embryos, the gene is a simple transcriptional unit containing two exons totalling 1347 nucleotides (nt) and a single small 5' intron of 54 nt. Compared to the enzyme from microorganisms, the deduced DHOdehase protein of 405 amino acids shows strong similarities within the presumptive catalytic portions of the protein. However, the N-terminal portions of these proteins are highly dissimilar, presumably reflecting diversity in the intracellular localization of DHOdehase in the different organisms. The Drosophila melanogaster protein contains N-terminal sequences that are typical of other mitochondrial intermembrane space proteins in animal cells.