The biliary metabolites of 17 alpha-ethynyl[3H]estradiol (3H-EE2) and their enterohepatic circulation were studied in female rats. Rapid and extensive elimination of radioactivity in bile followed i.v. administration of 3H-EE2. The metabolites consisted largely of beta-glucuronides of 2-hydroxy-EE2 and, to a lesser extent, 2-methoxy-EE2. Intraduodenally infused radiolabelled biliary metabolites underwent enterohepatic circulation; 15 +/- 6% (mean +/- S.D., n = 4) of the 3H was excreted in the bile of the recipient rats in 5.5 h. The metabolites excreted by the recipients were qualitatively and quantitatively similar to the infused metabolites. The 1(4)-glutathione adduct of 2-hydroxy-EE2 was synthesized, but was not observed in either the donors' or recipients' bile.