During mammalian hibernation, metabolic rate can be reduced to <5% of the euthermic rate as a result of coordinated suppression of multiple energy expensive metabolic processes. Gene transcription is one of these and the present study examines mechanisms of transcriptional control that could contribute to lowering the rate of gene expression in torpor. Histone deacetylases (HDAC) have been linked to gene silencing and measured HDAC activity was 1.82-fold higher in skeletal muscle of hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus, compared with euthermic controls. Western blotting also showed that HDAC1 and HDAC4 protein levels were 1.21-and 1.48-fold higher, respectively, in muscle from torpid animals. Histone H3 was also evaluated by Western blotting. Total histone H3 was unchanged but two forms of covalently modified histone H3 that are associated with active transcription (phosphorylated Ser 10 and acetylated Lys 23) were significantly reduced by 38-39% in muscle during hibernation. Finally, RNA polymerase II activity was measured using a PCR-based approach; activity in muscle from hibernating squirrels was only 57% of the euthermic value. These data support an overall decrease in transcriptional activity in skeletal muscle of hibernating animals that is accomplished by multiple molecular mechanisms.