Two forms of chicken vasoactive intestinal polypeptide (VIP) mRNA have been identified by reverse transcription (RT)-PCR and RNase protection assay. The shorter form of chicken VIP mRNA encodes a protein that does not contain an analogue of rat peptide histidine isoleucine (PHI) 1-27 or human peptide histidine methionine 1-27. The larger form encodes both VIP and a chicken analogue of PHI 1-27 in the same protein product. Three VIP cDNAs isolated from a chicken hypothalamic cDNA library were derived from the shorter mRNA. Sequence analysis of the longest clone identified an open reading frame that codes for a 165 amino acid preproVIP protein and contains two polyadenylation signals. In situ hybridisation with an oligonucleotide probe from the VIP cDNA sequence showed that VIP-encoding mRNA occurs in cells in the basal hypothalamus, an area of the brain known to contain VIP neurosecretory neurones. RT-PCR of total RNA from liver, kidney, gut, pancreas, pituitary, cerebellum, forebrain and hypothalamus, using primers derived from the VIP cDNA sequence, showed that the shorter form of VIP mRNA is present in all of these tissues. The sequence of the longer form of VIP mRNA was obtained by sequencing a portion of the VIP gene from genomic DNA. This revealed a potential exon that was not represented in the VIP cDNA clones analysed. RT-PCR with primers from this sequence showed that it was expressed in the gut and hypothalamus. RNase protection assays confirmed the presence of the two forms of mRNA in gut and hypothalamus. The relative proportions of the two mRNA forms were: 97.8% VIP only, 2.2% PHI/VIP in the hypothalamus and 98.5% VIP only, 1.5% PHI/VIP in the gut. In conclusion, chicken VIP mRNA is alternatively spliced. The shortest form, which encodes a preproprotein containing only the VIP peptide, is the most abundant. The longer form of chicken VIP mRNA encodes a preproprotein containing sequences for both VIP and a chicken form of PHI.