Somatostatin (SRIH) regulates pituitary adrenocorticotropin (ACTH) secretion by interacting with a family of homologous G protein-coupled membrane receptors. The SRIH receptor subtypes (sst 1–sst 5) that control ACTH release remain unknown. Using novel, subtype-selective SRIH analogs, we have identified the SRIH receptor subtypes involved in regulating ACTH release from AtT-20 cells, a model for cell line pituitary corticotropes. Radioligand-binding studies with 125 I-SRIH-14 and 125 I-SRIH-28 showed that SRIH-14 and SRIH-28 recognized specific, high-affinity and saturable membrane-binding sites. Nonpeptidyl agonists with selectivity for the sst 2 (L-779,976; compound 2) or sst 1/sst 5)(L-817,818; compound 5) receptor subtypes potently displaced 125 I-SRIH-28 from AtT-20 cell membranes, while agonists selective for the sst 1 (L-779,591; compound 1), sst 3 (L-796,778; compound 3) or sst 4 (L-803,087; compound 4) subtypes were inactive. Tyr 11-SRIH-14, compound 2 (sst 2) or compound 5 (sst 5) inhibited forskolin and corticotropin-releasing hormone (CRH)-induced increases in intracellular cAMP. Furthermore, the sst 2 and sst 5 agonists potently inhibited CRH-induced ACTH release from AtT-20 cells. These results provide the first evidence that sst 2 and sst 5 receptor subtypes, but not sst 1, sst 3 or sst 4, inhibit cAMP accumulation and regulate ACTH secretion in the AtT-20 cell model of the rodent corticotrope.