The Drosophila melanogaster Prat gene encodes amidophosphoribosyltransferase (PRAT; EC 2.4.2.14), which performs the first step in de novo purine nucleotide synthesis. Prat mutations have a recessive lethal phenotype that is found for other genes encoding enzymes in this pathway. The D. melanogaster genome project has revealed a second gene, CG10078 or Prat2, encoding a protein with 76% amino acid sequence identity with Prat. The two genes map to different arms of chromosome 3 and have different intron/exon organizations, as we confirmed by cDNA sequence analysis of Prat2. With the goal to determine the functional significance of this gene duplication, we isolated and sequenced two PRAT-encoding genes from Drosophila virilis. We find that the two D. virilis genes are orthologous to the two D. melanogaster genes in terms of intron/exon organization, amino acid coding sequence, and 5' noncoding sequence. The absence of introns in both DmelPrat and DvirPrat genes suggests that Prat originated from a retrotransposition of Prat2 and that the gene duplication has been preserved in the two species since their divergence approximately 40 million years ago. Analysis of mRNA expression in development shows that maternal expression, detected in adult ovaries and embryos prior to the onset of zygotic transcription, is present for Prat but not Prat2 in both species. Taken together, these findings support the notion that two PRAT-encoding genes have evolved distinct functions in both Drosophila species.