The biosynthesis of TRH in hypophysiotropic neurons of the paraventricular nucleus (PVN) is inversely regulated by feedback effects of circulating levels of thyroid hormones. As the PVN contains little or no deiodinase activity, the enzyme necessary to convert T4 to biologically active T3, we determined whether feedback inhibition of pro-TRH mRNA in thyroid hormone-sensitive neurons of the PVN is mediated exclusively by circulating levels of T3. The concentration of pro-TRH mRNA in the PVN of hypothyroid male rats receiving constant infusions of T3 over 7 days from ip implanted osmotic minipumps was studied by in situ hybridization histochemistry using computerized image analysis. Pro-TRH mRNA could not be suppressed to euthyroid levels by an infusion of T3 that returned plasma T3 levels to normal and required the infusion of higher concentrations of T3 that elevated plasma T3 into the supranormal range. By regression analysis, the mean concentration of plasma T3 required to suppress pro-TRH mRNA to euthyroid levels was estimated to be 110.3 ng/dl, similar to the amount of T3 estimated to be necessary to suppress TSH secretion from the anterior pituitary (108.7 ng/dl). We conclude that both T3 and T4 contribute to feedback inhibition of TRH biosynthesis in hypophysiotropic neurons of the PVN and propose that the effects of T4 on the PVN could be mediated after its monodeiodination at a different locus within the brain.