A peptide which relaxes rat uterine smooth muscle and exhibits homology with the mammalian C-type natriuretic peptide (CNP) has previously been identified in platypus (Ornithorhynchus anatinus) venom from its partial N-terminal amino acid sequence. In this study we describe the purification, detailed structure, synthesis and pharmacological characteristics of this peptide, which has been designated ovCNP-39 (Ornithorhynchus venom C-type natriuretic peptide). Elucidation of the 39-residue amino acid sequence confirms the homology with mammalian CNPs. These peptides produce hypotension in vivo and relax smooth muscle in vitro, but are poorly characterised in terms of physiological function. ovCNP-39 is equipotent with human/rat/porcine CNP-22 in eliciting cyclic guanosine 5'-monophosphate (cGMP) elevation in cultured vascular smooth muscle cells, suggesting that, like CNP, it acts through the ANPB natriuretic peptide receptor subtype. The direct elevation of cGMP in vascular smooth muscle by ovCNP-39 may underlie the vasodilatory effects of platypus envenomation.