Vasoactive intestinal polypeptide (VIP) is a 28-amino-acid hormone produced primarily by neural tissues. The amino acid sequence of VIP is similar to that of a number of gastrointestinal hormones, including glucagon and secretin. VIP is synthesized as part of a polyprotein, pre-proVIP, which generates, in addition to VIP, an additional bioactive peptide known as PHM. As a first step toward understanding the molecular basis of pre-proVIP gene expression, we have isolated the pre-proVIP gene and have determined its structure. The gene is approximately 9 kb long and is interrupted by six introns which appear to divide the gene into functional domains. One of the introns occurs within the 3'-untranslated region of the gene.