Thyroid hormone is important in the regulation of synthesis and secretion of thyroid-stimulating hormone (TSH) in the anterior pituitary, but its role in the control of hypothalamic thyrotropin-releasing hormone (TRH) is controversial. To determine whether thyroid hormone regulates the function of TRH in the hypothalamic tuberoinfundibular system, a study was made of the effect of hypothyroidism on thyrotropin-releasing hormone messenger RNA (proTRH mRNA) and TRH prohormone in the rat paraventricular nucleus. Extracts of rat hypothalamic paraventricular nucleus were examined by quantitative Northern blot analysis, and coronal sections of rat brain were examined by in situ hybridization histochemistry and immunocytochemistry. A nearly twofold increase in proTRH mRNA was observed in hypothyroid animals; this increase could be obliterated by levothyroxine treatment, suggesting an inverse relation between circulating thyroid hormone and proTRH mRNA. In situ hybridization showed that this response occurred exclusively in medial parvocellular neurons of the paraventricular nucleus. A simultaneous increase in proTRH mRNA and immunoreactive TRH prohormone in this region suggests that hypothyroidism induces both transcription and translation of the TRH prohormone in the paraventricular nucleus.