The larvae of Gynaephora groenlandica, a long-lived moth endemic to the high arctic, are perennially freeze-tolerant and able to increase their freeze-tolerance by synthesizing glycerol. Cold-induced mitochondrial changes were correlated (using electron microscopy, DNA staining, cytochrome c assay, and oxygen uptake) with glycerol production (using NMR spectroscopy) in larvae under different acclimations and in the field. Hypometabolism in summer- or warm-acclimated larvae led to glycerol accumulation. Extended exposure to near-zero or freezing temperatures caused mitochondrial degradation and glycerol accumulation. Rapid freezing of warm-acclimated larvae did not result in mitochondrial breakdown. Mitochondrial reconstitution upon warm-acclimation occurred much more rapidly (less than 1 week) than did degradation (greater than 2 months). Concomitant with mitochondrial breakdown was reduced oxidative metabolism, but the cytochrome c concentration remained independent of acclimation temperature. The adaptive response to cold by mitochondrial degradation and glycerol accumulation by G. groenlandica may be linked to diapause in other species of ectotherms.