The western painted turtle Chrysemys picta bellii can survive without oxygen (anoxia) for months while overwintering, during which time metabolic rate must be decreased so that ATP demand does not exceed anoxic ATP supply. In order to understand how metabolic depression is achieved in non-excitable turtle cells, we have optimized a hepatocyte cell culture system. We are approaching control of hepatocyte metabolic depression from multiple intersecting avenues, including mitochondrial signaling, actin and tubulin cycling, and mitochondrial dynamics. Actin and tubulin cycling are large consumers of cellular ATP pools, and we hypothesized that their cycling activity would be downregulated by overwintering conditions. We observed inhibition of cytoskeletal dynamics at overwintering temperature (4°C), signaling that is not induced by acute administration of either cyanide or oxygen tensions down to 0.1 Torr. A lesser response was observed with chronic exposure to cyanide. Acute decrease in temperature caused inhibition of actin polymerization, while cortical actin partially depolymerized. Hepatocytes plated at 4°C failed to reorganize the actin and tubulin cytoskeleton and mitochondrial density was low. Acute or chronic low temperature, or chronic cyanide exposure, resulted in reduced 2D cellular surface area. Increase in cytosolic calcium and associated mitochondrial depolarization appear key to actin cycling arrest.