Considerable attention has been focused on both highly oxidized low-density lipoprotein (ox-LDL) and mildly oxidized LDL (mox-LDL) as important risk factors for cardiovascular disease. Further, 5-hydroxytryptamine (5-HT) appears to play a crucial role in the development of atherosclerotic plaque. We assessed the interaction of oxidatively modified LDL and its major oxidative components, ie, hydrogen peroxide (H₂O ₂), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) with 5-HT on DNA synthesis in vascular smooth muscle cells (VSMCs). Growth-arrested rabbit VSMCs were incubated in serum-free medium with native LDL, mox-LDL, ox-LDL (all 50 ng/mL), H₂O₂ (0.5 μM), LPC (1 μM), or HNE (0.1 μM) for 24 hours followed by 5-HT (5 μM) for another 24 hours. DNA synthesis in VSMCs was measured by [³H]thymidine incorporation. Significant effects on [³H]thymidine incorporation were observed in VSMCs incubated with mox-LDL (129%), ox-LDL (129%), H₂O₂ (119%), LPC (115%), HNE (127%), or 5-HT (183%) in contrast with native LDL (113%). The mitogenic effect of 5-HT was potentiated by mox-LDL, ox-LDL, H₂O₂, LPC, or HNE (183 to 365%, 274%, 304%, 339%, or 273%, respectively) but not by native LDL (240%). The mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (10 μM) signficantly inhibited the mitogenic effect of 5-HT but did not influence the effects of mox-LDL, ox-LDL, H₂O₂, LPC, or HNE. The intracellular antioxidant N-acetylcysteine (400 μM) siginificantly inhibited the mitogenic effects of mox-LDL, ox-LDL, H₂O₂, LPC, and HNE but not that of 5-HT. Our results suggest that mox-LDL, ox-LDL, and their major components H₂O ₂, LPC, and HNE act synergistically with 5-HT in inducing VSMC DNA synthesis via MAPK and redox-sensitive pathways, contributing to the development of atherosclerotic plaque.