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 thyroid-stimulating hormone (TSH). Administration of thyrotropin-releasing hormone (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 prepro-TRH and 5'-deiodinase type II (5'-DII) in brain sections of six rats sleep deprived for 16-21 days, when there was marked hypothyroxinemia, and in sections from animals yoked to the experimental protocol as well as from sham controls. TRH transcript levels in the paraventricular nucleus (PVN) were essentially unchanged at 15-16 days but increased to about threefold control levels in three of four rats sleep deprived for 20-21 days, a change comparable to that typically found in prolonged experimental hypothyroidism. There was no evidence for suppression of 5'-DII 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 prepro-TRH mRNA expression indicates that alterations in posttranscriptional stages of TRH synthesis, processing, or release likely mediate the central hypothyroidism induced by sleep deprivation.