The somatostatin (somatotropin release-inhibiting factor; SRIF) receptor subtypes sst_2A and sst₅ are frequently co-expressed in SRIF-responsive cells, including endocrine pituitary cells. We previously demonstrated that sst_2A and sst₅ exhibit different sub-cellular localizations and regulation of cell surface expression, although they have similar signaling properties. We investigated here whether sst_2A and sst₅ functionally interact in cells co-expressing the two receptor subtypes. We stimulated both transfected cells stably expressing sst_2A alone (CHO-sst_2A) or together with sst₅ (CHO-sst_2A+5) and the pituitary cell line AtT20, which endogenously expresses the two receptor subtypes, with either the non-selective agonist [D-Trp⁸]-SRIF-14 or the sst₂-selective agonist L-779,976. In CHO-sst_2A cells, stimulation with either ligand resulted in the loss of approximately 75% of cell surface SRIF binding sites and massive internalization of sst_2A receptors. The cells were desensitized to subsequent stimulation with [D-Trp⁸]-SRIF-14 which failed to inhibit forskolin-evoked cAMP accumulation. Similarly, in CHO-sst_2A+5 and AtT20 cells [D-Trp⁸]-SRIF-14 induced the loss of 60-70% of SRIF binding sites as well as massive sst_2A endocytosis. By contrast, in cells expressing both sst_2A and sst₅, selective stimulation of sst_2A with L-779,976 resulted only in 20-40% loss of cell surface binding and markedly reduced sst_2A internalization. Consequently, whereas CHO-sst_2A+5 and AtT20 cells stimulated with [D-Trp⁸]-SRIF-14 were desensitized to a second stimulation with the same agonist, cells pre-stimulated with L-779,976 were not desensitized to subsequent [D-Trp⁸]-SRIF-14 stimulation. These findings indicate that the presence of sst₅ in the same cells modulates trafficking and cell surface regulation of sst_2A and cellular desensitization to the effects of SRIF.