Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) was investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitive internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST. Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala1-SST-14 remains inside the cell. Delineation of sequence motifs responsible for internalization of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser-346, Ser-351, and Thr-357) within the carboxy-terminal tail of which Ser-351 and Thr-357 were the most effective ones. Chimeras in which various segments of the carboxyl terminus of SSTR4 were replaced by the corresponding regions of SSTR3 were internalized as long as they contain the Ser/Thr motif. However, this internalization reaction was suppressed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control element in that region. Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr-Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subtype.