Steps 6 and 7 of de novo purine synthesis are performed by 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide synthetase (SAICARs), respectively. In vertebrates, a single gene encodes AIRc-SAICARs with domains homologous to Escherichia coli PurE and PurC. We have isolated an AIRc-SAICARs cDNA from Drosophila melanogaster via functional complementation with an E. coli purC purine auxotroph. This cDNA encodes AIRc yet is unable to complement an E. coli purE mutant, suggesting functional differences between Drosophila and E. coli AIRc. In vertebrates, the AIRc-SAICARs gene shares a promoter region with the gene encoding phosphoribosylamidotransferase, which performs the first step in de novo purine synthesis. In Drosophila, the AIRc-SAICARs gene maps to section 11B4-14 of the X chromosome, while the phosphoribosylamidotransferase gene (Prat) maps to chromosome 3; thus, the close linkage of these two genes is not conserved in flies. Three EMS-induced X-linked adenine auxotrophic mutations, ade4(1), ade5(1), and ade5(2), were isolated. Two gamma-radiation-induced (ade5(3) and ade5(4)) and three hybrid dysgenesis-induced (ade5(5), ade5(6), and ade5(8)) alleles were also isolated. Characterization of the auxotrophy and the finding that the hybrid dysgenesis-induced mutations all harbor P transposon sequences within the AIRc-SAICARs gene show that ade5 encodes AIRc-SAICARs.