GH3 cells express receptors for the neuropeptide somatostatin (SRIF). In the present study, we have identified and characterized SRIF1 and SRIF2 receptors in GH3 cells using the radioligands [125I]MK 678 and [125I]CGP 23996. [125I]MK 678 binding to SRIF1 receptors was saturable and of high affinity and was potently inhibited by SRIF analogs with a rank order of potency of MK 678 > SRIF > SRIF 28 > CGP 23996. [125I]CGP 23996 binding to SRIF2 receptors was also saturable and of high affinity, and was potently inhibited by SRIF analogs with a rank order of potency of SRIF 28 > SRIF > CGP 23996, but was not inhibited by MK 678. Agonist pretreatment of GH3 cells differentially regulated SRIF1 and SRIF2 receptors. [125I]MK 678 binding to SRIF1 receptors was readily diminished after pre-exposure of GH3 cells to SRIF or MK 678. [125I]CGP 23996 binding to SRIF2 receptors was unaffected by pretreatment with MK 678 and was only partially affected by pretreatment with SRIF. [125I]MK 678 binding to SRIF1 receptors was abolished in the presence of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thio)triphosphate, but [125I]CGP 23996 binding to SRIF2 receptors was unaffected. The SRIF1 receptor mediates inhibition of adenylyl cyclase activity, as SRIF and MK 678 inhibited forskolin-stimulated cyclic AMP accumulation in these cells to the same extent. GH3 cells are a unique model system for investigations of the pharmacological, biochemical and functional properties of these two receptor subclasses.