The receptor-mediated activation of phospholipase D (PLD) is a major signaling pathway in several cell systems. This study determined the effects of epidermal growth factor (EGF) on PLD activity in normal rat osteoblastic cells. Primary cultures were obtained from fetal rat calvaria by sequential collagenase digestion and seeded in BGJb media supplemented with 10% fetal calf serum. PLD activity was assayed by the transphosphatidylation reaction in [H3]myristic acid (5 μCi/ml)-labeled cells treated with EGF in the presence of 5% ethanol and measuring the production of phosphatidylethanol (PEtOH). Lipids were extracted and separated by thin-layer chromatography, detected by iodine staining, and the areas of interest were scraped off and transferred to vials for scintillation counting. EGF significantly increased PEtOH production in a dose-dependent manner and at short (10-60 s) and long (up to 30 minutes) incubation periods (p < 0.05). Phosphatidic acid levels were also significantly increased (p < 0.05) compared with unstimulated controls, but the levels were ∼60% less than those of PEtOH. 4b-phorbol 12-myristate, 13-acetate (PMA) also produced a significant increase in PEtOH levels when compared with unstimulated control cultures, but when PMA was added together with EGF, the production of PEtOH was reduced about 30%. Pretreatment of cells with the protein kinase C (PKC) inhibitor H-7 caused a significant increase in PEtOH levels, compared with cells stimulated with EGF alone. Preincubation of cells with pertussis toxin produced a partial decrease in PEtOH levels. This study demonstrates that EGF activates the PLD signaling cascade in normal rat osteoblastic cells and that the pathway appears to involve, at least in part, a PKC- and Gi protein-dependent mechanism.