PTH, a major regulator of bone remodeling and a therapeutically effective bone anabolic agent, stimulates several signaling pathways in osteoblastic cells. Our recent studies have revealed that PTH activates phospholipase D (PLD) -mediated phospholipid hydrolysis through a RhoA-dependent mechanism in osteoblastic cells, raising the question of the upstream link to the PTH receptor. In the current study, we investigated the role of heterotrimeric G proteins in mediating PTH-stimulated PLD activity in UMR-106 osteoblastic cells. Transfection with antagonist minigenes coding for small peptide antagonists to G alpha 12 and G alpha13 subunits of heterotrimeric G proteins prevented PTH-stimulated activation of PLD, whereas an antagonist minigene to G alphas failed to produce this effect. Effects of pharmacological inhibitors (protein kinase inhibitor, Clostridium botulinum exoenzyme C3) were consistent with a role of Rho small G proteins, but not of cAMP, in the effect of PTH on PLD. Expression of constitutively active G alpha12 and G alpha13 activated PLD, an effect that was inhibited by dominant-negative RhoA. The results identify G alpha12 and G alpha13 as upstream transducers of PTH effects on PLD, mediated through RhoA in osteoblastic cells.