Five somatostatin receptors (SSTRs) bind somatostatin-14 (S-14) and somatostatin-28 (S-28) but SSTR-5 has the highest affinity for S-28. To determine whether S-28 acting through SSTR-5 mediates inhibition of glucagon-like peptide-1 (GLP-1) fetal rat intestinal cell cultures were treated with somatostatin analogs with relatively high specificity for SSTRs 2-5. S-28 dose-dependently inhibited GLP-1 secretion stimulated by gastrin-releasing peptide more potently than S-14 (EC₅₀: 0.01 vs.5.8 nM). GLP-1 secretion was inhibited by an SSTR-5 analog BIM-23268 more potently than S-14 and nearly as effectively as S-28. The SSTR-5 analog L-372,588 also suppressed GLP-1 secretion equivalent to S-28 but a structurally similar peptide L-362,855 (Tyr to Phe at position 7) was ineffective. An SSTR-2 selective analog was less effective than S-28 and an SSTR-3 analog was inactive. Separate treatment with GLP-1-(7-36)NH₂ increased S-28 and S-14 secretion by 3- and 5-fold; BIM-23268 abolished S-28 without altering S-14. The results indicate that somatostatin regulation of GLP-1 secretion occurs via S-28 through activation of SSTR-5. GLP-1-stimulated S-28 secretion is also autoregulated by SSTR-5 activation suggesting a feed-back loop between GLP-1 and S-28 modulated by SSTR-5.