Skeletal muscle hexokinase (HK) from Richardson's ground squirrels was analyzed to determine how the enzyme is regulated during hibernation, a state of cold torpor. The HK II isozyme dominated in muscle and ∼15% of total HK was bound to the insoluble fraction. HK maximum activity was 33% lower in hibernator muscle and the enzyme showed a significantly higher Km ATP (by 80%) and a lower Ki for glucose-6-P (by 40%) than euthermic HK (assayed at 22°C). However, 5°C assay significantly reduced Km glucose of hibernator HK. Stimulation of AMP-dependent protein kinase (AMPK) in hibernator extracts elevated the HK activity and reduced Km ATP, but did not affect euthermic HK. Stimulation of protein phosphatases significantly lowered the HK activity in both situations. AMPK-dependent phosphorylation was confirmed by immunopreciptiation of 32P-labeled HK. DEAE-Sephadex ion exchange chromatography revealed two peaks of HK in hibernator muscle extracts (low and high phosphate forms), whereas only a single peak of phospho-HK was present in euthermic muscle. We conclude that differential control of muscle HK in euthermic versus hibernating states is derived from two main regulatory influences, reversible protein phosphorylation and temperature effects on kinetic properties.