Human estrogen receptors alpha and beta (ERalpha and ERbeta) greatly differ in their target genes, transcriptional potency and cofactor-binding capacity, and are differentially expressed in various tissues. In classical estrogen response element (ERE)-mediated transactivation, ERbeta has a markedly reduced activation potential compared with ERalpha; the mechanism underlying this difference is unclear. Here, we report that the binding of steroid receptor coactivator-1 (SRC-1) to the AF-1 domain of ERalpha is essential but not sufficient to facilitate synergy between the AF-1 and AF-2 domains, which is required for a full agonistic response to estradiol (E2). Complete synergy is achieved through the distinct hinge domain of ERalpha, which enables combined action of the AF-1 and AF-2 domains. AF-1 of ERbeta lacks the capacity to interact with SRC-1, which prevents hinge-mediated synergy between AF-1 and AF-2, thereby explaining the reduced E2-mediated transactivation of ERbeta. Transactivation of ERbeta by E2 requires only the AF-2 domain. A weak agonistic response to tamoxifen occurs for ERalpha, but not for ERbeta, and depends on AF-1 and the hinge-region domain of ERalpha.