Internalization of G protein-coupled receptors and desensitization of the hormonal responses are well characterized in vitro for several hormonal systems. The physiological role of internalization for a G protein-coupled receptor involved in homeostatic functions has not been established; although it has been assumed based on in vitro data. We have previously shown that phosphorylation of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor is required for its internalization and for the desensitization of the responsiveness to PTH and PTHrP in vitro; the internalization and desensitization response is impaired in a PTH/PTHrP receptor mutant bearing serine to alanine mutations in the phosphate acceptor sites. To understand the physiological role of receptor internalization on calcium homeostasis we have knocked-in the internalization-impaired PTH/PTHrP receptor mutant using homologous recombination technology. The genetically modified animals exhibited calcium levels no different from control animals; but PTH levels were one third of those in control animals indicating that homeostasis could be maintained only by 3 fold suppression of PTH secretion. We also analyzed the calcemic response to PTH in vivo. Here we show that mice expressing the internalization-impaired PTH/PTHrP receptor mutant have dramatically exaggerated cAMP and calcemic responses to subcutaneous PTH administration when compared with control animals given the same dose. These data show for the first time the role of G protein receptor phosphorylation and internalization per se in the regulatory function of an endocrine system controlled by a G protein-coupled receptor.