The role of glutathione and the response of components of the ascorbic acid-glutathione cycle in cold acclimation and the acquired freezing tolerance of jack pine (Pinus banksiana Lamb.) seedlings were investigated. An increase in the reduced to oxidized glutathione mole ratio was correlated with the increase in root soluble and membrane-bound protein thiol concentrations during cold acclimation and after a freeze and thaw event. All the enzymes involved in the ascorbic acid-glutathione cycle were regulated by low temperatures and increased activities of ascorbic peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase were observed after the conditioning of the seedlings to low temperatures. Our results suggest that these enzymes play a protective role following the exposure of the seedlings to freezing temperatures.