Induction of cyclin D1 gene transcription by estrogen receptor α (ERα) plays an important role in estrogen-mediated proliferation. There is no classical estrogen response element in the cyclin D1 promoter, and induction by ERα has been mapped to an alternative response element, a cyclic AMP-response element at -57, with possible participation of an activating protein-1 site at -954. The action of ERβ at the cyclin D1 promoter is unknown, although evidence suggests that ERβ may inhibit the proliferative action of ERα. We examined the response of cyclin D1 promoter constructs by luciferase assay and the response of the endogenous protein by Western blot in HeLa cells transiently expressing ERα, ERαK206A (a derivative that is superactive at alternative response elements), or ERβ. In each case, ER activation at the cyclin D1 promoter is mediated by both the cyclic AMP-response element and the activating protein-1 site, which play partly redundant roles. The activation by ERβ occurs only with antiestrogens. Estrogens, which activate cyclin D1 gene expression with ERα, inhibit expression with ERβ. Strikingly, the presnce of ERβ completely inhibits cyclin D1 gene activation by estrogen and ERα or even by estrogen and the superactive ERαK206A. The observation of the opposing action and dominance of ERβ over ERα in activation of cyclin D1 gene expression has implications for the postulated role of ERβ as a modulator of the proliferative effects of estrogen.