Endocytosis of somatostatin receptors could regulate cellular responses to the two natural peptides, somatostatin-14 and somatostatin-28, and to synthetic ligands used in the clinical diagnosis and symptomatic therapy of neuroendocrine tumours. The five cloned SSTRs with or without epitope tags at their carboxyl-termini were expressed in rat insulinoma 1046-38 cells. Application of the two natural peptides or octreotide, at 37 degrees C but not at 4 degrees C, to cells transfected with somatostatin receptor subtype 2 or 3 cDNA resulted in a significant decrease of cell surface binding-sites for 125I-Tyr11-somatostatin-14. In contrast, cells transfected with subtype 5 cDNA only responded to stimulation with octreotide or somatostatin-28. Cells transfected with subtype 1 cDNA responded to somatostatin-14 and 28, while cells expressing subtype 4 cDNA showed no response. Confocal microscopy revealed that 6 min after stimulation with somatostatin-14 at 37 degrees C, tagged somatostatin receptor subtypes 1, 2 and 3 were internalized into vesicles. Internalization was not observed at 4 degrees C in the presence of 0.4 M sucrose and 80 microM phenylarsine oxide and hence proceeded via endocytosis through clathrin-coated pits and vesicles. After 20 min the internalized receptors appeared in perinuclear vesicles and after 120 min they reappeared at the plasma membrane. This recycling was not sensitive to cycloheximide and, hence, not dependent on de novo protein synthesis. Recovery of cell surface receptors was, however, inhibited by brefeldin A, monensin and bafilomycin A1, indicating that receptor recycling proceeded through vesicular traffic of acidified compartments. The data are consistent with the assumption that the observed agonist and subtype specific internalization of somatostatin receptors in a neuroendocrine cell line may be important for tumour diagnosis and therapy and, thus, suggest a manifold control in cellular signalling.