Vascular smooth muscle cell (VSMC) proliferation induced by various growth factors has been implicated in a wide variety of pathological processes including hypertension, atherosclerosis and restenosis after angioplasty. We investigated the interactions among well-known potent vasoconstrictor substances, endothelin-1 (ET-1), angiotensin II (Ang II), and serotonin (5-HT) on VSMC proliferation. Growth-arrested rabbit VSMCs were incubated with different concentrations of ET-1 in the absence or presence of Ang II, 5-HT, or both. VSMC proliferation was examined by increases in [3H]thymidine incorporation into DNA and cell number. ET-1, Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner. ET-1 had a maximal effect at a concentration of 0.5 μM (259% of control), Ang II at 1 μM (173%), and 5-HT at 50 μM (205%). When added together, ET-1 (0.1 μM) and Ang II (1 μM) synergistically induced DNA synthesis (341%). When the vasoconstrictors were tested in combination, even non-mitogenic concentrations of ET-1 (0.01 nM) potentiated 5-HT (5 μM)-induced DNA synthesis (404%). Coincubation of ET-1 (0.01 μM) with Ang II (1 μM) and 5-HT (5 μM) synergistically induced DNA synthesis (566%). These effects on DNA synthesis were paralleled by an increase in cell number. The ETA/B non-selective receptor antagonist, TAK044 (1 μM) and the ETA receptor antagonist, BQ123 (1 μM), but not the ETB receptor antagonist, BQ788 (1 μM), inhibited the mitogenic effect of ET-1 and its interaction with Ang II or 5-HT. In addition, TAK044 (1 μM) or BQ123 (1 μM) along with the AT1 receptor antagonist, candesartan (1 μM), the 5-HT2A receptor antagonist, sarpogrelate (10 μM), or both, inhibited the interactions of ET-1 with Ang II or 5-HT. Our results suggest that Ang II and 5-HT could potentiate ET-1-induced VSMC proliferation. Inhibition of ETA, AT1, and 5-HT2A may be effective in the treatment of VSMC proliferative disorders associated with hypertension, atherosclerosis and restenosis after angioplasty.