An in vitro xanthine/xanthine oxidase reaction system was used to generate superoxide anions that significantly stimulated tritiated [3H]thymidine incorporation into endothelium-removed (denuded) male rat aortic explants. Tritiated thymidine uptake was used as an index of vascular smooth-muscle cell (VSMC) proliferation. Superoxide dismutase (SOD) significantly attenuated the oxygen free radical-induced proliferative response of these cells. 17β-estradiol (17β-E) significantly inhibited superoxide anion-induced VSMC proliferation. In contrast, the growth-modifying effects of 17β-E were not mimicked by 17α-estradiol (17α-E), progesterone, or testosterone. The pure estrogen receptor (ER) antagonist, ICI 164,384, reversed the growth-inhibitory effect of 17β-E. 17β-Estradiol failed directly to reduce in vitro superoxide anion production or to modify xanthine oxidase activity. Therefore, these data indicate that 17β-E, through an ER-dependent mechanism, specifically and significantly inhibited superoxide anion-mediated SMC proliferation in denuded rat aortic explants.