The somatostatin [somatotropin release-inhibiting factor (SRIF)] receptor subtypes sst(2A) and sst(5) are frequently coexpressed in SRIF-responsive cells, including endocrine pituitary cells. We previously demonstrated that sst(2A) and sst(5) exhibit different subcellular localizations and regulation of cell surface expression, although they have similar signaling properties. We investigated here whether sst(2A) and sst(5) functionally interact in cells coexpressing the two receptor subtypes. We stimulated both transfected cells stably expressing sst(2A) alone (CHO-sst(2A)) or together with sst(5) (CHO-sst(2A+5)) and the pituitary cell line AtT20, which endogenously expresses the two receptor subtypes, with either the nonselective agonist [D-Trp(8)]-SRIF-14 or the sst(2)-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-Trp(8)]-SRIF-14, which failed to inhibit forskolin-evoked cAMP accumulation. Similarly, in CHO-sst(2A+5) and AtT20 cells, [D-Trp(8)]-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 sst(5), selective stimulation of sst(2A) with L-779,976 resulted in only 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-Trp(8)]-SRIF-14 were desensitized to a second stimulation with the same agonist, cells prestimulated with L-779,976 were not desensitized to subsequent [D-Trp(8)]-SRIF-14 stimulation. These findings indicate that the presence of sst(5) in the same cells modulates trafficking and cell surface regulation of sst(2A) and cellular desensitization to the effects of SRIF.