Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [(3)H]-labeled E(2) or E(1), and with [(14)C]-E(1) in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E(2) right harpoon over left harpoon E(1)) by trophoblast varied with development; it favored E(2) in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [(3)H](2)O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E(2) in both free and sulfate fractions. It was concluded that local metabolism of E(2) is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.