Natriuretic peptides (NPs) play important roles in osmoregulatory and cardiovascular systems of vertebrates. For functional studies of NPs, rainbow trout (Oncorhynchus mykiss), a euryhaline fish, is an interesting model. The information on homologous NPs of salmonid fish is, however, still incomplete with respect to C-type NP (CNP). In this study, we isolated cDNAs encoding the precursor of CNP from the brain of trout. Predicted mature CNP (CNP-22) sequence was identical to that of killifish Fundulus heteroclitus, and only one amino acid was different from that of the eel Anguilla japonica, demonstrating a greater conservation among different teleost species than is found with atrial NP (ANP) and ventricular NP (VNP). While the preprosegment of trout CNP retained 57% similarity to the eel sequence, similarities were low to those of sharks and tetrapods. The major site of expression identified by RT-PCR was the brain with minor expression in the atrium. The putative mature CNP-22 was synthesized and its biological activity was compared with other trout NPs (ANP and VNP) using trout ventral aorta, efferent branchial and celiacomesenteric arteries and anterior cardinal vein in vitro. Synthetic trout CNP-22 relaxed all pre-contracted vessels with potencies comparable to trout ANP and VNP.