Drosophila melanogaster genomic DNA spanning an adenosine2 gene rearrangement breakpoint (in cytological map region 26B1-2) was cloned and a composite ade2 base sequence was derived from this DNA and from a corresponding cDNA. Based on genetic evidence, the ade2 gene is thought to encode the purine biosynthetic enzyme formylglycineamide ribotide amidotransferase (FGARAT). The cDNA hybridizes to a 4.8-kb message that encodes a 1354 amino acid polypeptide with extensive similarity to Escherichia coli FGARAT. The D. melanogaster FGARAT amino acid sequence is considerably more like that of the E. coli enzyme than is the FGARAT of B. subtilis, which has two polypeptides with, collectively, 969 amino acids. It is suggested that the taxonomically anomalous similarity between the eukaryotic FGARAT and that from E. coli may indicate horizontal exchange of genetic material. On the basis of substantially greater conservation of sequences shared by all three species compared with those present only in E. coli and D. melanogaster, we suggest that no radical alteration of enzymatic function accompanied the transition between the single-gene and the two-gene state.