Retinoids are multipotent modulators of cellular functions and suppress cytokine-induced production of nitric oxide (NO) in several cell types. We have explored the mechanisms by which retinoic acid (RA) regulates NO production in rat aortic smooth muscle cells (VSMC), which express NOS2 in response to proinflammatory cytokines. RA inhibited interleukin-1 beta (IL-1 beta)-induced NOS2 mRNA expression and NO production. These effects were attenuated by the retinoic acid receptor (RAR) antagonist CD3106, indicating that they were mediated through retinoic acid receptors (RARs). The synthetic retinoid agonists CD336 (which specifically binds RAR alpha) and CD367 (which binds all RARs) but not agonists specific for RAR beta, RAR gamma, or RXRs reduced IL-1 beta-induced NOS2 expression and NO production. When transfecting VSMC with a 1570-bp NOS2 promoter fragment fused to a luciferase reporter gene, the NOS2 promoter activity was inhibited by Rk These results indicate that retinoids modulate NO production in VSMC via RAR alpha, which inhibits the transcription of the NOS2 gene.