Somatostatin-14 (S-14) and somatostatin-28 (S-28) bind to five distinct membrane receptors (SSTRs), but S-28 has higher affinity for SSTR-5. Whether S-28 acting through SSTR-5 regulates inhibition of peptide YY (PYY) secretion was tested in fetal rat intestinal cell cultures. S-28 and S-14 caused dose-dependent inhibition of PYY secretion stimulated by gastrin-releasing peptide, but S-28 was more potent than S-14 (EC₅₀ 0.04 vs. 13.2 nM). PYY was inhibited by two analogs with affinity for SSTR-5, BIM-23268 and BIM-23052, more potently than S-14 and as effectively as S-28. The SSTR-5 analog L-362855 suppressed PYY equivalent only to S-14, but the structurally related peptide L-372588 (Phe to Tyr at position 2) was equipotent to S-28, whereas L-372587 (Phe to Tyr at position 7) caused no inhibition. An SSTR-2 analog decreased PYY secretion similar to S-14, and an SSTR-3 analog was ineffective. PYY secretion stimulated by phorbol 12-myristate 13-acetate and by forskolin was also more potently suppressed by S-28 and the octapeptide SSTR-5 analogs. The results indicate that S-28 mediates inhibition of gastrin-releasing peptide-stimulated PYY secretion through activation of SSTR-5 and includes suppression of cAMP- and protein kinase C-dependent pathways. Substitution of a single hydroxyl group confers differences in SSTR-5 agonist properties, suggesting region specificity for the intrinsic activity of this receptor subtype.