Recent evidence indicates that phosphatidylcholine breakdown by phospholipase D (PLD) is an important cellular control mechanism. We investigated the signaling pathway participating in prostaglandin E2 (PGE2)-induced PLD activation in osteoblast-like MC3T3-E1 cells. PGE2 stimulated PLD activity, as measured by choline generated from phosphatidylcholine, just after the stimulation. The reaction reached a plateau 15 minutes later. PGE2 stimulated PLD activity in a dose-related manner and also increased inositol phosphate (IP) formation. However, the EC50 value for PGE2-induced IP formation is lower than that for PLD activation. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, stimulated PLD activity, and a combination of PGE2 and TPA potentiated it in an additive manner. Although NaF, a heterotrimeric GTP-binding protein activator, significantly stimulated PLD activity, this effect was not augmented by combination with PGE2. PGE2-induced PLD activity was markedly suppressed by either chelating extracellular Ca2+ by EGTA or pertussis toxin. These findings suggest that osteoblasts might have at least two PLD activation mechanisms which involve PKC-dependent or -independent pathways. However, present results indicate that PKC is unlikely to be essential to PGE2-induced PLD activation. On the contrary, pertussis toxin-sensitive GTP-binding protein and extracellular Ca2+ might play important roles in the pathway of PGE2-induced PLD activation.