Thyroxine (T4) uptake from amniotic fluid was investigated in fetal sheep. Samples of fetal and maternal blood and of amniotic fluid were obtained from indwelling catheters at specific intervals after intra-amniotic injection of T4. T4 and reverse T3 (rT3) were measured by radioimmunoassay. Basal levels of T4 were 7.3 +/- 0.92, less than 2, and 6.28 +/- 0.49 microgram/dl in the fetus, amniotic fluid, and ewe, respectively. Basal levels of rT3 were 3,858 +/- 214, 189 +/- 62, and 385 +/- 20 pg/ml in the fetus, amniotic fluid, and ewe, respectively. T4 and rT3 rose progressively in the fetus with maximum concentrations of 25 to 30 microgram/dl T4 by 10 hours and 11,000 to 14,000 pg/ml rT3 by 20 hours after intra-amniotic injection of 500 microgram of T4. These concentrations returned toward baseline by 50 and 70 hours for T4 and rT3, respectively. The increase of fetal T4 was proportional to the amount of T4 injected in a range of 250 and 2,500 microgram. Esophageal ligation abolished the changes in fetal T4 but not rT3. Amniotic fluid rT3 increased with time after intra-amniotic injection of T4 and returned to baseline long after amniotic fluid T4 had reached basal levels. This pattern persisted despite esophageal ligation. T4 was converted to rT3 during incubation in amniotic fluid in vitro. It is concluded that (1) substantial amounts of T4 are taken up by fetal fetal sheep from the amniotic fluid by deglutition, (2) increases in fetal concentrations of T4 and rT3 are related to the amount of T4 added to the amniotic fluid, (3) amniotic fluid and fetal rT3 concentrations increase following intra-amniotic injection of T4 in the absence of significant uptake of T4 by the fetus, and (4) significant amounts of T4 may be converted to rT3 in vitro during incubation of T4 in amniotic fluid.