The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor=SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst 3), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst 3 receptors; [ 125I]LTT-SRIF 28 ([Leu 8, d-Trp 22, 125I-Tyr 25]-SRIF 28), [ 125I]Tyr 10-cortistatin, [ 125I]CGP 23996, and [ 125I]Tyr 3-octreotide labelled the fsst 3 receptor with high affinity (p K d values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different B max values; 4500, 4000, 3400 and 1500 fmol/mg, respectively. The affinities of SRIF peptides and analogues determined for fsst 3 receptors displayed the following rank order of potency: seglitide=SRIF 25>SRIF 14=SRIF 28>cortistatin 14>BIM 23014>RC160=L361,301=octreotide≥BIM 23052≥L362,855>CGP23996>BIM 23056>BIM 23030=cycloantagonist>SRIF 22. The pharmacological profiles determined with [ 125I]LTT-SRIF 28, [ 125I]CGP 23996 and [ 125I]Tyr 10-cortistatin correlated highly significantly ( r=0.96–0.99), whereas [ 125I]Tyr 3-octreotide binding was rather divergent ( r=0.78–0.81). Further, [ 125I]Tyr 3-octreotide- and [ 125I]CGP 23996-labelled sites showed higher affinity for the various peptides than [ 125I]LTT-SRIF 28 and [ 125I]Tyr 10-cortistatin-labelled sites, although there were exceptions. [ 125I]LTT-SRIF 28-binding to fsst 3 receptors and human sst 1–5 receptors was compared; the fsst 3 binding profile correlated better with the hsst 5- than with the hsst 3 receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst 3 transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fsst 3 receptor to G i α and/or G o α . [ 125I]LTT-SRIF 28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [ 125I]LTT-SRIF 28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst 3 receptor, in agreement with expression of the fsst 3 receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst 3 receptor has a pharmacological profile compatible with that of a SRIF 1 receptor, although the rank order of affinity of fsst 3 is closer to that of hsst 5 than hsst 3 receptors, as may be found when comparing very distantly related species. The fsst 3 receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian sst 3 receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst 3 receptor in brain as well as other yet to be defined SRIF receptor subtypes.