A heterogeneous population of cDNAs (designated Vupur3) encoding phosphoribosylglycinamide formyltransferase (GART; EC 2.1.2.2) was isolated from a cowpea (Vigna unguiculata L. Walp.) nodule library. Three classes of cDNA with the same ORF, but differing in their 3′-UTRs, were identified. Southern analysis and sequencing of genomic DNA confirmed that these differences result from alternative splicing of the primary transcript of a single Vupur3 gene. Alternative splicing does not appear to play a role in the production of soybean (Glycine max Merrill.) pur3 transcripts. The presence of the protein product of the Vupur3 gene, GART, in plastids and mitochondria was confirmed by immunoblotting with antibodies raised against the recombinant protein. The antibodies recognised two proteins with apparent molecular masses of 27 and 27.5 kDa in both mitochondria and plastids. All Vupur3 transcripts have two in-frame start codons that are active in wheatgerm in vitro transcription / translation experiments suggesting a mechanism by which the gene product could be targeted to two organelles. Like other genes encoding enzymes for purine synthesis, Vupur3 is expressed in nodules before nitrogen fixation begins but in contrast to these genes its expression does not increase markedly after nitrogen fixation begins.