In order to systematically investigate differences in gene expression between sleep and waking, mRNA differential display was used to examine mRNAs from the cerebral cortex of rats who had been spontaneously asleep, spontaneously awake, or sleep deprived for a period of 3 h. It was found that, while the vast majority of transcripts were expressed at the same level among these three conditions, the expression of a subset of mRNAs was modulated by sleep and waking. Half of these transcripts had known sequences in databases. RNAs expressed at higher levels during waking included those for the transcription factors c-fos, NGFI-A, and rlf, as well as three transcripts encoded by the mitochondrial genome, those for subunit I of cytochrome c oxidase, subunit 2 of NADH dehydrogenase, and 12S rRNA. As shown by in situ hybridization, the level of RNAs encoded by the mitochondrial genome was uniformly higher during waking in many cortical regions and in several extracortical structures. By contrast, mRNA levels corresponding to two mitochondrial protein subunits encoded by the nuclear genome were unchanged. This finding suggests the hypothesis that an increase in the level of mitochondrial RNAs may represent a rapid regulatory response of neural tissue to adapt to the increased metabolic demand of waking with respect to sleep.