Most loss of function mutations of GNAS identified in different forms of pseudohypoparathyroidism disrupt not only the stimulatory G protein α-subunit (Gsα), but also its paternally expressed variant, XLαs. However, the possibility that XLαs deficiency contributes to disease pathogenesis has remained unexplored. We therefore examined the signaling property of human XLαs and the effects of one novel (XLαsH704P or GsαH362P) and two previously described (XLαsDelI724 and XLαsY733X or GsαDelI382 and GsαY391X, respectively) GNAS mutations on either XLαs or Gsα activity. Confocal immunofluorescence microscopy detected human XLαs immunoreactivity at the plasma membrane of transduced mouse embryonic fibroblasts null for endogenous Gsα and XLαs (GnasE2−/E2− cells). Cholera toxin- and isoproterenol-induced cAMP accumulation in GnasE2−/E2− cells transiently expressing wild-type human XLαs was similar to that in cells transiently expressing wild-type Gsα. Human XLαs, like Gsα, mediated PTH-induced cAMP accumulation in GnasE2−/E2− cells coexpressing PTH receptor type 1 and either of these proteins. Moreover, overexpression of human XLαs or Gsα markedly enhanced the PTH-induced cAMP accumulation in opossum kidney cells that endogenously express PTH receptor type 1. In contrast, each XLαs mutant failed to mediate isoproterenol- and PTH-induced cAMP accumulation in transduced GnasE2−/E2− cells. XLαsDelI724 showed a reduced cholera toxin response over the basal level compared with wild-type XLαs, and XLαsH704P completely failed to respond to cholera toxin. These findings were comparable to those observed with each corresponding Gsα mutant transiently expressed in GnasE2−/E2− cells. Thus, mutations that typically inactivate Gsα also impair XLαs activity, consistent with a possible role for XLαs deficiency in diseases caused by paternal GNAS mutations.