We have previously reported the participation of MAP-, Rho- and phosphoinositide-3 (PI3) kinases in separate pathways in serotonin (5-HT)-induced proliferation of pulmonary artery smooth muscle cells (SMCs). In this study we investigated the possible participation of PLD and phosphatidic acid (PA) in this growth process. 5-HT stimulated a time-dependent increase in [3H]-phosphatidyl-butanol and phosphatidic acid (PA) generation. Exposure of SMCs to 1-butanol or overexpression of an inactive mutant of human PLD1R898R blocked 5-HT-induced proliferation. Furthermore, 1-butanol inhibited 5-HT activation of S6K1 and S6 protein, downstream effectors of mTOR, by 80 % and 72%, respectively, and partially blocked activation of ERK by 30%, but had no effect on other associated signaling pathways. Exogenous PA caused cellular proliferation and revitalized cyclin D1 expression by 5-HT of the 1-butanol-treated cells. PA also reproduced activations by 5-HT of mTOR, S6K1 and ERK. Transfection with inactive human PLD1 reduced 5-HT-induced activation of S6K1 by about 50%. Inhibition of 5-HTR 2A with ketaserin blocked PLD activation by 5-HT. Inhibition with PI3K inhibitor failed to block either activation of PLD by 5-HT or PA dependent S6K1 phosphorylation. Taken together, these results indicate that ligation of the 5-HTR 2A by 5-HT initiates PLD activation in SMCs, and that its product, PA, is an early signaling molecule in 5-HT-induced pulmonary artery SMC proliferation. Signaling by PA produces its downstream effects primarily through the mTOR/ S6K1 pathway and to a lesser extent through the ERK pathway. Hydrolysis of cell membrane lipid may be important in vascular effects of 5-HT.