We examined the effect of platelet-derived growth factor (PDGF) on the activation of phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. PDGF-BB stimulated both the formation of choline (EC50 = 15 ng/ml) and inositol phosphates (EC50 = 5 ng/ml). However, PDGF-BB had little effect on the formation of phosphocholine. The formation of choline stimulated by a combination of PDGF-BB and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C (PKC)-activating phorbol ester, was additive. H-7, an inhibitor of protein kinases, inhibited 12-O-tetradecanoylphorbol-13-acetate-induced choline formation, whereas HA1004, a control for H-7 as PKC inhibitor, had little effect. Neither H-7 nor HA1004 affected the PDGF-BB-induced formation of choline. Genistein and methyl 2,5-dihydroxycinnamate, inhibitors of protein tyrosine kinases, dose dependently inhibited the PDGF-BB-induced formation of choline. PDGF-BB stimulated Ca2+ influx from extracellular space. PDGF-BB-induced choline formation was significantly reduced by chelating extracellular Ca2+ with EGTA. PDGF-BB stimulated DNA synthesis of MC3YT3-E1 cells, and H-7 inhibited the DNA synthesis. These results strongly suggest that PDGF activates phosphatidyl-choline-hydrolyzing phospholipase D independently from PKC activated by phosphoinositide hydrolysis in osteoblast-like cells, and that both tyrosine kinase activation and Ca2+ influx are essential for this mechanism.