A single dose of tritiated estradiol-17 beta (3H-E2 beta) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 +/- 2.1 (mean +/- S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding 14C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of 3H-E2 beta in plasma were 10.9 +/- 1.9 min and 118 +/- 18 ml/min/kg body weight, respectively. The calculated production rate of E2 beta at 19.2 hr before oviposition was 19.5 +/- 5.7 ng/min based on the plasma level (93 +/- 22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 +/- 0.6 min post injection were, in descending order: estradiol-17 beta-3-sulfate (E2 beta-3S: 14.9 +/- 2.7), estradiol-17 alpha-3-sulfate (E2 alpha-3S; 5.7 +/- 0.3), estrone (E1; 4.6 +/- 0.5), estrone sulfate (E1S; 2.2 +/- 0.5), and estradiol-17 alpha (E2 alpha; 1.2 +/- 0.4). As time proceeded, the relative concentration of E2 alpha-3S gradually increased so that by 43.2 +/- 1.0 min it became the most abundant identifiable metabolite (12.3 +/- 1.1) followed by E2 beta-3S (9.1 +/- 1.7), E1S (1.2 +/- 0.6), E1 (0.7 +/- 0.4) and E2 alpha (0.3 +/- 0.2). These findings are consistent with the view that one of the major pathways of E2 beta metabolism in the circulation of the hen is via E2 beta in equilibrium E2 beta-3S in equilibrium E1S in equilibrium E2 alpha-3S.