Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PL), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study we investigated the role of key enzymes involved in PL metabolism: the PL-hydrolyzing enzymes phospholipase A2 (PLA₂), and the PL-synthesizing enzyme lyso-phosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic smooth muscle cells (BASMC). Concomitantly with inducing BASMC proliferation, thrombin activates cytosolic PLA₂ (cPLA₂), expressed by selective release of arachidonic acid (AA) and mRNA expression, as well as LPAAT, expressed by non-selective incorporation of fatty acid, and mRNA expression. Specific inhibitors of these enzymes, AACOCF₃ for cPLA₂ and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA₂ and LPAAT activation in thrombin-induced smooth muscle cell proliferation. Thrombin acts through the proteolysis-activated receptor (PAR-1), and accordingly we found that the thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent the thrombin-induced activation of cPLA2 and LPAAT, implying that activation of cPLA₂ and LPAAT is required but not sufficient for thrombin-induced proliferation of BASMC.