Background—Cyclooxygenase (COX)-2 contributes to vascular smooth muscle cell (VSMC) proliferation induced by tumor necrosis factor (TNF) and angiotensin II. The present study demonstrates, however, that depending on prevailing conditions, COX-2–derived prostanoids may also inhibit VSMC proliferation.Methods and Results—TNF-&agr; stimulated proliferation of VSMCs by shortening the G1phase of the cell cycle. This effect was abolished by NS-398, a selective COX-2 inhibitor. Addition of TNF did not affect the protein-to-DNA ratio, measured by flow cytometry, suggesting that TNF does not induce VSMC hypertrophy. Inhibition of nitric oxide synthase (NOS) activity attenuated TNF-mediated increases in prostaglandin (PG) I2synthesis, whereas thromboxane (TX) A2production and COX-2 protein expression were unaffected. Moreover, inhibition of NOS activity increased TNF-mediated proliferation by ≈23%. Thus, NO preferentially stimulates PGI2production, suggesting that production of NO by VSMCs challenged with TNF limits the ability of the cytokine to increase proliferation. NO donors increased COX-2 protein expression and PGI2synthesis, had no effect on TXA2production, and decreased cell numbers by 50%, indicating that expression of COX-2 per se might not be sufficient to support proliferation. The effects of NO donors were prevented when COX-2 activity was inhibited with NS-398.Conclusions—The COX-2–dependent proliferative response of VSMCs to TNF was modulated in an NO-dependent manner, and PGI2derived from COX-2 might contribute to the antiproliferative effect of NO donors.