To understand the role of growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) and to examine the functional significance of the co-expression of GHRH and PACAP in fish, their receptors were characterized in zebrafish. Three cDNAs encoding the PAC1 receptor, the VPAC1 receptor, and the partial GHRH receptor were identified from zebrafish. Functional expression of the PAC1 and VPAC1 receptors revealed that both are potently coupled to the adenylyl cyclase pathway, but only the PAC1 receptor is coupled to the phospholipase C pathway. Transcripts for all three receptors were widely distributed, often in an overlapping pattern in the adult zebrafish. Also, one splice variant of the partial GHRH receptor and three splice variants of the PAC1 receptor were identified from adult zebrafish. The long GHRH receptor transcript contained a 27 amino acid insert in transmembrane domain 5 encoding a premature stop codon leading to a truncated receptor protein. For the PAC1 receptor, two of the splice variants corresponded to the hop1 and hop2 variants characterized in mammals. The third splice variant identified from the gill encoded a novel 107 bp insert containing a premature stop codon. Therefore, PACAP and GHRH have widespread, overlapping target sites suggesting a coordinated role for these hormones in evolution.