Upon agonist exposure, most membrane receptors internalize into the cell as part of an adaptation process. Receptor domains that mediate internalization have been defined for several receptor classes, e.g. growth factor and transport receptors, but not yet for any of the numerous members of the family of G protein-coupled receptors (GPCRs), having seven putative transmembrane helices. With the use of deletion mutations, we previously showed that a small region in the middle of the third intracellular loop (i3) appears to be required for the agonist-induced internalization of the Hm1 muscarinic cholinergic receptor (Lameh, J., Philip, M., Sharma, Y. K., Moro, O., Ramachandran, J., and Sadée, W. (1992) J. Biol. Chem. 267, 13406-13412). Using point mutations, we now demonstrate that domain 286-292 (ESLTSSE) is required, with TSS playing a crucial role. Although the i3 loops of GPCRs share minimal overall sequence identity, even among closely related subtypes, small domains containing multiple S/T residues are also present in several other GPCRs, e.g. peptide, catechol, and all the muscarinic cholinergic receptor subtypes. S/T-->A mutations in analogous i3 domains also prevented receptor internalization in the case of Hm3 and attenuated internalization of Hm2. We propose that an S/T-rich domain in the i3 loop, possibly via phosphorylation reactions, regulates one pathway of GPCR internalization.