The human estrogen receptors (ERs) α and β interact with 17β-estradiol (17β-E2), estrone, 17α-estradiol, and the ring B unsaturated estrogens, equilin, 17β-dihydroequilin, 17α-dihydroequilin, equilenin, 17β-dihydroequilenin, 17α-dihydroequilenin, Δ8-estrone, and Δ8, 17β-E2 with varying affinities. In comparison to 17β-E2, the relative binding affinities of most ring B unsaturated estrogens were 2- to 8-fold lower for ERα and ERβ, however, some of these unique estrogens had two to four times greater affinity for ERβ than ERα. The transcriptional activity of these estrogens in HepG2 cells transfected with ERα or ERβ, or both, and the secreted-alkaline phosphatase gene showed that all estrogens were functionally active. 17β-E2 induced the activity of secreted-alkaline phosphatase by ERα to a level higher than any other estrogen. Activity of other estrogens was 12–17% that of 17β-E2. In contrast, 17β-E2 stimulated the activity of ERβ to a 5-fold lower level than that with ERα, whereas the activity of other estrogens was 66–290% that of 17β-E2, with equilenin being the most active. The presence of both ER subtypes did not alter the functional activity of 17β-E2, although it further enhanced the activity of 17β-dihydroequilin (200%), 17β-dihydroequilenin (160%), and Δ8, 17β-E2 (130%). Except for 17β-E2, no correlation was observed between the functional activities and their binding affinities for ER. In conclusion, our results show that the effects of ring B unsaturated estrogens are mainly mediated via ERβ and that the presence of both ER subtypes further enhances their activity. It is now possible to develop hormone replacement therapy using selective ring B unsaturated estrogens for target tissues where ERβ is the predominant ER.