Somatostatin (SS) is a widely distributed polypeptide that exerts inhibitory effects on hormone secretion and cell proliferation by interacting with five different receptors (SST1-SST5). Beta-arrestins have been implicated in regulating SST internalization, but the structural domains mediating this effect are largely unknown. The aim of this study was to characterize the intracellular mechanisms responsible for internalization of human SST5 in the rat pituitary cell line GH3 and to identify the SST5 structural domains involved in this process. To this purpose we evaluated, by fluorescence microscopy and biochemical assay, the ability of wild-type, progressive C-terminal truncated and third cytoplasmatic loop mutants SST5-DsRed to associate with beta-arrestin-enhanced green fluorescent protein and to internalize under SS28 stimulation. The truncated mutants were comparable to the wild-type receptor with respect to recruitment of beta-arrestin-2 and internalization, whereas the third loop mutants R240W, S242A, and T247A showed the abolishment or reduction of arrestin association and a significant reduction of receptor internalization (14.4%, 29%, and 30.9% vs. 52.4% of wild type) and serine phosphorylation upon SS28 stimulation. Moreover, we evaluated the ability of simultaneous mutation of these three residues (R240, S242, and T247) and C-terminal truncated receptors to internalize. The progressive truncation of the C-terminal tail resulted in a progressive increased internalization (21.6%, 36.7%, and 41%, respectively) with respect to the full-length total third-loop mutant (15%). In conclusion, our results indicate the SST5 third intracellular loop as an important mediator of beta-arrestin/receptor interaction and receptor internalization, whereas they suggest that residues 328-347 within the C terminus may play an inhibitory role in receptor internalization.