In human and rat, tissue-specific proteolytic processing of identical proglucagon precursors yield tissue-specific proglucagon-derived peptides. In contrast, in many non-mammalian vertebrates alternative mRNA splicing yields different proglucagon precursors in different tissues. Thus alternative mRNA splicing, in part, limits the choices of proglucagon-derived peptides that can be produced by proteolytic processing. Stomach proglucagon mRNAs from the rainbow trout and Xenopus laevis were found not to encode the proglucagon-derived peptide glucagon-like peptide 2 (GLP-2). To determine if the absence of GLP-2 was a general feature of stomach proglucagons we isolated and characterized proglucagon cDNAs from the stomach and the pancreas of the dog, a mammal that expresses the proglucagon gene in the stomach. A major proglucagon transcript of about 1100 bases and a minor transcript of about 800 bases were identified in both stomach and pancreas. The coding sequences of both the stomach and pancreatic proglucagon transcripts were identical. Therefore, tissue-specific proteolytic processing, and not alternative mRNA splicing, must regulate the production of tissue-specific proglucagon-derived peptides from the stomach of the dog.