Sleep deprivation in rats results in progressive declines in circulating concentrations of both total and free thyroxine (T4) and triiodothyronine (T3) without an expected increase in plasma TSH. Administration of TRH results in appropriate increases in plasma TSH, free T4 and free T3 across experimental days, suggesting deficient endogenous TRH production and/or release. This study examined transcriptional responses related to TRH regulation following sleep deprivation. In situ hybridization was used to detect and quantitate expression of mRNAs encoding preproTRH and 5'-deiodinase type II (5'D-II) in brain sections of 6 rats sleep deprived for 16 to 21 days, when there was marked hypothyroxinemia, and in sections from animals yoked to the experimental protocol as well as sham controls. TRH transcript levels in the paraventricular nucleus were essentially unchanged at 15 to 16 days but increased to approximately 3-fold control levels in 3 of 4 rats sleep deprived for 20 to 21 days, a change comparable to that typically found in prolonged experimental hypothyroidism. There was no evidence for suppression of 5'D-II mRNA levels, which would be a sign of T3 feedback downregulation of neurons in the PVN. A failure to increase serum TSH in response to hypothyroxinemia and to increased preproTRH mRNA expression indicates that alterations in posttranscriptional stages of TRH synthesis, processing, or release likely mediate the central hypothyroidism induced by sleep deprivation.