Monocyte chemotactic protein 1 (MCP-1), which is synthesized by vascular cells, is a chemoattractant for monocytes and has been implicated in a wide range of acute and chronic inflammatory processes characterized by monocyte infiltration, including atherosclerosis. However, it is unclear whether MCP-1 is able to modulate vascular smooth muscle cell (VSMC) proliferation. We assessed the effect of MCP-1 on VSMC proliferation and its interaction with serotonin (5-HT), a mitogen for VSMCs. Growth-arrested VSMCs were stimulated with different concentrations of MCP-1 (25–200 ng/ml) and 5-HT (5 and 50 µ M) in serum-free medium. DNA synthesis in VSMCs was measured by [<sup>3</sup>H]thymidine incorporation. 5-HT at concentrations of 5 and 50 µ M significantly stimulated DNA synthesis by 1.8- and 2.1-fold over the control value, respectively (p < 0.0001). However, MCP-1 at the concentrations tested did not have any significant effect on DNA synthesis. Even though MCP-1 (50 ng/ml) by itself is not mitogenic, when added to 5-HT, it significantly amplified the mitogenic effect of 5-HT compared with that of 5-HT alone (p < 0.0001). The 5-HT<sub>2A</sub> receptor antagonist sarpogrelate (10 µ M) and its major metabolite M-1 (0.1 µ M), pertussis toxin (10 ng/ml), Src family protein tyrosine kinase (PTK) inhibitor PP2 (1 µ M), protein kinase C (PKC) inhibitor Ro31-8220 (0.1 µ M) and mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 (10 µ M) significantly inhibited the mitogenic effect of 5-HT and its interaction with MCP-1. Anti-MCP-1 antibody (2 µg/ml) and the Janus kinase 2 (JAK2) inhibitor AG490 (10 µ M) significantly inhibited the interaction of MCP-1 with 5-HT. Further, the amplified mitogenic effect of 5-HT with MCP-1 was completely reversed by the combined use of sarpogrelate with anti-MCP-1 antibody. Our results suggest that MCP-1 amplifies the mitogenic effect of 5-HT on VSMCs. The mitogenic effect of 5-HT may be mediated by the G protein-Src family PTK-PKC-MAPK pathway. The activation of the JAK2/signal transducer and activator of transcription 3 pathway by MCP-1 in addition to the MAPK pathway by 5-HT may explain the potentiating effect of MCP-1 on 5-HT-induced mitogenesis.