The changing roles of the hypothalamus and pituitary in regulating thyroid hormone levels in the rat during ontogeny has not been fully elucidated. It has been reported that endogenous TRH begins to stimulate TSH secretion at 5–8 days after birth but that the pituitary responds to hypothyroidism during late gestation. To determine the onset and extent of TRH response to low thyroid hormone levels during ontogeny, normal and hypothyroid rats treated with methimazole for 7 days were sacrificed at 16 days gestation (E16), 20 days gestation (E20), 7, 21 and 56 days after birth (n = 5/study group). Plasma hormones were assayed from pregnant mothers, pups (pooled) and adults. Levels of TRH mRNA were measured in the paraventricular nuclei (PVN) by in situ hybridization histochemistry. A labeled 48-base cDNA oligonucleotide for TRH was hybridized with brain slices (n = 6/animal) in the region of the medial parvocellular division of the PVN of the hypothalamus and the signal was quantitated by digitized computer analysis. Plasma-free T4 levels decreased and plasma TSH levels increased in the animals treated with methimazole as compared to the euthyroid controls. TRH mRNA was detected in the PVN at E16 after brain slices were dipped in emulsion and granules observed by dark-field microscopy. In the euthyroid animals, TRH mRNA increased from E20 (150 ± 9 OD units) to 7 days (222 ± 5 OD units) and remained unchanged at 21 days (252 ± 27 OD units) and 56 days (244 ± 6 OD units). The hypothyroid rats as compared to age-matched controls, had TRH mRNA levels that were unchanged at E16 and E20 and increased to 121% at 7 days (269 ± 9 0D units; p < 0.001), 176% at 21 days (461 ± 26 OD units; p < 0.001), and 225% at 56 days (545 ± 20 OD units; p < 0.001). In summary, TRH mRNA was present in the PVN at E16 and increased until 7 days after birth. TRH mRNA was not altered with hypothyroidism until after E20 and prior to or on the 7th day after birth when levels increased in response to low thyroid hormone levels. Thus, the ontogeny of the regulation of thyroid hormone feedback on TRH mRNA levels and of TSH secretion by endogenous TRH may be temporally associated suggesting an important role of PVN maturation in the development of the thyr