To better understand the evolution of pituitary adenylate cyclase-activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH), we isolated the cDNAs encoding these peptides from the brains of five species of fish: sturgeon, whitefish, grayling, flounder and halibut. Both hormones are encoded in tandem in full-length cDNAs. We compared the phylogenetic relationship among these and other known sequences encoding PACAP. In closely related species, transcripts encoding PACAP and GHRH are strongly conserved in the hormone coding regions, moderately conserved in the signal peptide, cryptic peptide and 3'-untranslated regions, but are most varied in the 5'-untranslated regions.Next, we compared the deduced amino acid sequences for the peptides to known sequences. Sturgeon and whitefish have a PACAP(38) peptide sequence that is 92% conserved compared to human PACAP(38), the highest for a fish reported to date. GHRH is the lesser conserved of the two peptides with only 39% to 45% conservation between fish and human.Each of the five fish species had a second cDNA encoding a short precursor lacking GHRH(1-32), the bioactive portion of GHRH. This suggests that exon skipping in GHRH-PACAP transcripts may be an important mechanism for regulating the ratio of PACAP to GHRH peptides.