To investigate the rT3 effect on iodothyronine-5'-deiodinating activity (I-5'-DA) in cultured fetal mouse liver, liver on the 19th day of gestation, in which little or no I-5'-DA was detected and, therefore, rT3 was very stable, was cultured in medium containing thyroid hormone-depleted fetal calf serum supplemented with cortisol, insulin, and various concentrations of iodothyronines. After 4-15 days of culture, I-5'-DA in the homogenate was assessed by the amount of iodide released from outer ring-labeled rT3 and expressed as picomoles of 127I- per mg protein/min. I-5'-DA was induced by T3 (10(-9)-10(-8) M) and T4 (10(-7) M). In contrast, rT3 could not induce I-5'-DA at 10(-8)-10(-6) M. Furthermore, rT3 significantly decreased I-5'-DA induced by T3 (10(-8) M) at almost equimolar concentrations (1-5 X 10(-8) M). The inhibitory action of rT3 was reversible and was specific for I-5'-DA; no alteration in malic enzyme activity or intracellular glutathione concentration was detected. The inhibitory effect of rT3 on I-5'-DA was dose dependent, and the enzyme activity decreased with a half-life of 16 h in the presence of 10(-6) M rT3. The inhibitory effect was not due to contaminating rT3 in the liver homogenates. Lineweaver-Burk analysis revealed that the decrease in I-5'-DA was due to a decrease in the maximum velocity, whereas no alteration in Km was detected, suggesting that rT3 decreases the amount of 5'-deiodinase induced by T3. The minimal free rT3 concentration capable of inhibiting the induction of iodothyronine-5'-deiodinase was approximately 10(-10) M, which may be attainable in vivo, as in amnionic fluid. In summary, we have demonstrated that rT3 exerts a strong antagonistic effect against T3 in inducing I-5'-DA in cultured fetal mouse liver.