Food deprivation in laboratory rats induces profound changes in the neuroendocrine system. We have investigated the hypothalamic and pituitary responses of the hypothalamo- pituitary thyroid axis to 48-h food deprivation in Sprague- Dawley rats. Peripheral T₃ and hypophysial portal TRH were measured by RIA, and TSHβ, PRL, and pro-TRH mRNA were measured using in situ hybridization histochemistry. Peripheral total T₃ was greatly reduced in food-deprived rats. Hypothalamic portal blood TRH levels declined significantly with time in control animals. The initial level of TRH in the portal blood of food-deprived rats was significantly reduced compared to that in controls, but did not fall further with time. In situ hybridization histochemistry revealed significantly lower pro-TRH mRNA in the paraventricular nucleus of food-deprived animals, while pro-TRH mRNA in the reticular nucleus remained unaltered. Furthermore, in the anterior pituitary, TSHβ mRNA decreased significantly in food-deprived animals, while PRL mRNA was unaltered. We conclude that the reduction in circulating T₃ after food deprivation appears to be due primarily to decreased hypothalamic TRH synthesis and release. (Endocrinology129: 2714–2718, 1991)