Enteric bacteria have been postulated to have a role in thyroid economy by promoting the hydrolysis of thyroid hormone conjugates of biliary origin, thus permitting the absorption and recycling of thyroxine (T4) and triiodothyronine (T3). An enterohepatic circulation of T3 might be more pronounced under conditions in which type I iodothyronine deiodinase activity (5'D-I) is inhibited, because this augments the accumulation of T3 sulfate conjugates in bile. This potential of increased gut reabsorption of T3 might explain, at least in part, the failure of serum T3 values to decrease appreciably when marked reductions in peripheral 5'D-I activity are induced by selenium deficiency or 6-anilino-2-thiouracil (ATU) administration. Thus, studies were performed to determine the effect of intestinal decontamination, in the absence and in the presence of 5'D-I inhibition, on plasma T4 and T3 concentrations. Groups of adult male rats received either enteric antibiotics or no antibiotics for 12 days and then, in half of the rats in each group, treatment for 10 days with ATU, a 5'D-I inhibitor that does not affect thyroid hormone synthesis. The activity of intestinal arylsulfatase and arylsulfotransferase, enzymes that catalyze hydrolysis of thyroid hormone conjugates, was reduced markedly by approximately 87% in rats that received antibiotics, regardless of whether or not they also received ATU. The ATU treatment markedly inhibited liver 5'D-I activity in antibiotic-treated as well as in non-antibiotic-treated rats (control = 399 +/- 32 U/mg protein (mean +/- SEM); ATU = 152 +/- 17: antibiotics = 351 +/- 29; antibiotics + ATU = 130 +/- 10; p < 0.01) and significantly increased plasma T4 and T3 sulfate (T4S, T3S) concentrations (control: T4S = 2.8 +/- 0.4 and T3S = 6.7 +/- 1.3 ng/dl; ATU: T4S = 6.2 +/- 1.4 and T3S = 10.6 +/- 2.1 ng/dl; antibiotics: T4S = 1.8 +/- 0.2 and T3S = 3.6 +/- 1.0 ng/dl; antibiotics + ATU: T4S = 6.8 +/- 0.7 and T3S = 9.7 +/- 1.8 ng/dl; p < 0.05). The ATU treatment was associated with a significant increase in plasma T4 and rT3 concentrations but did not affect plasma T3 concentrations, and intestinal decontamination did not alter these ATU-associated effects on circulating thyroid hormones. These results suggest that anaerobic enteric bacteria in the rat do not have an important role in recycling of thyroid hormones, either under normal conditions or in circumstances where 5'D-I activity is markedly reduced, and that increased gut absorption of T3 from T3S cannot explain the near-normal serum T3 values found when peripheral 5'D-I activity is markedly decreased.