To investigate the domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group-containing amino acids at the C-terminus. In mutant #2, we replaced threonines 392 and 399 in the third intracellular loop with glycines. Co-transfection of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhibition of PTH receptor signaling was associated with enhanced phosphorylation receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cAMP generation by the wild-type receptor and mutant #1, and caused phosphorylation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agonist-induced phosphorylation of mutant #2 compared with that of either the wild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was associated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 failed to cause receptor desensitization and inhibited receptor internalization. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intracellular loop are not required for agonist-induced phosphorylation of PTH receptors, but are critical for both agonist-induced internalization of PTH receptors and GRK2-mediated regulation of PTH receptor signaling.