Current heightened recognition of the importance of sulfated steroids led to the examination of conjugates in media from incubations of estrogens in tissues from the reproductive tract of stallions. Previously, we had reported a ‘new’ unidentified metabolite of estrone (E1) and [3H]-E1, located between 17β-estradiol (E2) and E1 reference standards on chromatography (HPLC) and identified tentatively as a stable 5α,6α-estrone epoxide. Stallion tissues were minced and incubated for 2 h with [3H]-E1 (1 × 106 cpm). Solid-phase extraction of unconjugated and conjugated steroids from media was followed by liquid scintillation counting (LSC), where radioactivity was mostly in the conjugate fractions (>80%). HPLC of conjugated steroids used an isocratic solvent system of acetonitrile/water (8:92) at 700 µL/min with detection by LSC. A radioactive peak between reference standards of E1 and E2 sulfates was examined, after solvolysis, in a second solvent system. Sulfated steroids yielded largely E1, whereas acid treatment of the unconjugated E1 epoxide had earlier formed 6α-OH-E1 almost exclusively. With sulfatase enzyme, at neutral pH, radioactivity was also seen mostly as E1 with trace amounts of polar material. Reduction with KBH4, however, led also to desulfation; radioactivity had alignment with E2 but even more had low retention times as for 6α/6β-OH-E2. These findings point to a different hydrolysis for desulfation; even more, they reveal an additional oxygen atom at C6 and are supportive of biological formation of 5α,6α-epoxides of E1 and E2. As possible alternatives to catechol estrogens, implicated in cancer, the ‘new’ estrogen metabolites and their sulfated forms may have special interest.