The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among somatostatin receptors in that it exhibits a very long intracellular carboxyl-terminal tail containing a large number of potential phosphate acceptor sites. Consequently, little is known about its agonist-dependent regulation. We have generated a series of phosphorylation-deficient mutants and determined crucial sites for its agonist-induced ß-arrestin mobilization, internalization and downregulation. Based on this information, we generated phosphosite-specific antibodies for carboxyl-terminal Ser337, Thr341 and Thr348 that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that pasireotide and octreotide promoted clearly less phosphorylation compared to the endogenous ligand somatostatin. Interestingly, the sst3-selective ligand L-796,778 did not promote any detectable phosphorylation or internalization. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. We also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1ß as key regulators of sst3 phosphorylation and dephosphorylation. Thus, we define the carboxyl-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, ß-arrestin recruitment and internalization.