Recent studies suggest that 2-methoxyestradiol (2-ME), an estrogen metabolite, has a similar inhibitory effect as 17-estradiol (E₂) on vascular tone. However, it is not known if 2-ME mediates the effects of E₂ or by what mechanism 2-ME regulates smooth muscle contraction. Therefore, we compared the effects of 2-ME and E₂ on rat aortic smooth muscle contraction. Pre-incubation with 2-ME (10 µM) for 1 h inhibited phenylephrine (PE)-induced tension in endothelium-intact, but not -denuded tissues, whereas E₂ inhibited PE-induced contraction in both preparations. The effects of 2-ME and E₂ on endothelium-intact preparations were prevented by L-NAME (NO synthase inhibitor). 2-ME treatment reduced PE-induced phosphorylation of the 20-kDa myosin regulatory light chain. The inhibitory effects of 2-ME and E₂ were not affected by ICI 182,780 (estrogen receptor antagonist) or actinomycin D (gene transcription inhibitor); however, the effect of 2-ME, but not that of E₂, was prevented by cycloheximide (protein synthesis inhibitor). Furthermore, the effect of E₂ was not blocked by 1-aminobenzotriazole (cytochrome P450 inhibitor) or Ro 41-0960 (catechol-O-methyltransferase inhibitor). The effect of 2-ME was not mimicked by microtubule-interfering agents (nocodazole or taxol). We conclude that 2-ME inhibits smooth muscle contractility through an endothelium- and NO-dependent mechanism, which does not involve estrogen receptors or microtubule disruption. The effect of 2-ME, but not E₂, involves de novo protein synthesis. 2-ME does not mediate the inhibitory effect of E₂ on smooth muscle contraction. These results support a potentially important role of 2-ME in the regulation of smooth muscle tone in the vasculature.