We have studied the structure and expression of the gene for vasoactive intestinal polypeptide (VIP) in rodents. We used a human cDNA to identify and clone a fragment of the rat VIP gene. This genomic fragment contained two separate exons, one encoding VIP itself and the other encoding a closely related neuropeptide, peptide histidine-isoleucine (PHI-27). Probes containing either exon, or both, hybridized to two messages: a prominent 1700-base (b) mRNA and a rare 1000-b species. These messages are expressed together in a tissue-specific manner, with highest levels in polyadenylated RNA from cerebral cortex and from small intestine, paralleling the reported levels of the neuropeptides themselves in these tissues. Using the rat genomic fragment as a probe, we isolated the mouse VIP gene in its entirety. The mouse gene is similar in organization to its human counterpart, with a total of 7 exons spanning 8 kilobases (kb). The 7th and largest exon, which is transcribed into the bulk of the 3' untranslated region of the messages, bears two potential polyadenylation sites 700 basepairs (bp) apart. S-1 nuclease protection with a fragment of this exon indicated that the two identifiable VIP messages differ in the extent of their 3' untranslated regions. Conversely, we found no evidence for differential splicing to produce messages encoding only one of the neuropeptides. Instead, specific oligonucleotide-directed digestion with RNase H demonstrated that all of the detectable mRNA from this gene contains both VIP and PHI coding sequences.