In this study, a somatostatin receptor (Sst) cDNA was cloned and sequenced from goldfish pituitary. The cDNA encodes a 390-amino acid type five-like Sst (designated as gfSst(5)). The amino acid sequence of the receptor has slightly higher homology to mammalian Sst(5'), compared with other mammalian Sst subtypes and recently identified fish Sst(1), Sst(2) and Sst(3). In CCL39-SRE-Luci cells stably expressing the cloned receptor, agonist radioligand [125I]LTT-SRIF(28'), a mammalian SRIF(28) analog, bound to a homogenous population of receptors with high affinity (nM K(d)). Competition binding studies showed that all three natural goldfish SRIF ligands, SRIF(14), [Pro(2)]SRIF(14), and goldfish SRIF(28) (gfSRIF(28)), and LTT-SRIF(28) bind the cloned gfSst(5) with high affinity and significantly stimulate [35S]GTPgammaS binding, with SRIF(28) peptides showing higher affinity in receptor binding and potency in [35S]GTPgammaS binding compared with SRIF(14) peptides. The receptor gene is highly and predominately expressed in pituitary; lower levels of the receptor mRNA were also detected in different brain regions by reverse transcriptor-polymerase chain reaction (RT-PCR) followed by Southern blot analysis.