The selectivity of G-protein recognition is determined by the intracellular loops (ICLs) of seven-transmembrane-spanning receptors. In a previous study, we have shown that the N-terminal and central portions of ICL2 from F525 to D530 participate in dual -/-protein activation by the thyroid-stimulating hormone receptor (TSHR). ICL3 is another major determinant for G-protein activation. Therefore, the aim of our study was to identify important amino acids within ICL3 of the TSHR to gain insight in more detail about its specific function for - and -protein activation and selectivity. Single-alanine substitutions of residues in the N-terminal, middle, and C-terminal region of ICL3 were generated. N-terminal residues Y605 and V608 and C-terminal positions K618, K621, and I622 were identified as selectively important for activation, whereas mutations in the center of ICL3 had no effect on TSHR signaling. Our findings provide evidence for an amino acid pattern in the N- and C-terminal part of ICL3, which is involved in -mediated signaling. Furthermore, molecular modeling of interaction of TSHR ICL2 and 3 with suggests three potential contact sites: TSHR C-terminal ICL3 with β5-6 loop of , TSHR ICL2 residues I523-R531 with β2-3 loop and N-terminal helix of , and TSHR ICL2/transmembrane helix (TMH) 3+ICL3/TMH6 with C-terminal tail of .