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In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca2+]i) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca2+]i increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca2+]i increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca2+]i increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-κB-driven luciferase activity in exogenously LPA1, LPA2, or LPA3-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA1, LPA2, and LPA3), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca2+]i increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.