The inhibitory effects of estrogen (17beta-estradiol) on atherosclerosis have been well documented in numerous animal models, and epidemiological evidence supports this protective effect in humans. The detailed mechanisms for this protection are not understood, but most are thought to be mediated through estrogen receptors (ERs), of which two are known (ERalpha and ERbeta). To investigate the role of ERalpha in the atheroprotective effect of 17beta-estradiol (E2), we ovariectomized female mice that lack apoE (AAee) or lack both apoE and ERalpha (alphaalphaee), and treated half of them with E2 for three months. E2 treatment of ovariectomized AAee females dramatically reduced the size of the lesions as well as their histological complexity. Plasma cholesterol was significantly reduced in this group, although the observed extent of protection by E2 was greater than could be explained solely by the change in lipid levels. In contrast, E2 treatment of ovariectomized alphaalphaee females caused minimal reduction in lesion size and no reduction in total plasma cholesterol compared with alphaalphaee mice without E2, demonstrating that ERalpha is a major mediator of the atheroprotective effect of E2. Nevertheless, E2 treatment significantly reduced the complexity of plaques in the alphaalphaee females, although not to the same degree as in AAee females, suggesting the existence of ERalpha-independent atheroprotective effects of E2.