1. The aim of these studies was to investigate a mitochondrial basis for changes in gluconeogenesis during hibernation. 2. State 3 respiration rates in liver mitochondria from hibernating ground squirrels were reduced by 62-66%. The limiting reaction appeared to be electron transport, particularly in respiratory complex III. 3. The mitochondrial ATP + ADP + AMP content was reduced by 29% during hibernation; cellular adenine nucleotide content was unchanged. 4. Pyruvate carboxylation in intact mitochondria was decreased 75% during hibernation, although total pyruvate carboxylase activity was not lower. 5. Rates of gluconeogenesis in intact hepatocytes isolated from hibernators were lower than in cells from non-hibernators.