A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3'-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin mu heavy chain are made from a single genetic locus.