Overwintering plants should survive the various biotic and abiotic stresses that occur during winter. Previous studies indicated that active oxygen species are involved in freezing, dehydration, anoxia and pathogen infections. As the importance of the events that occur in the intercellular compartment became apparent in disease resistance, we examined the nature of intercellular antioxidant enzymes in order to access their possible involvement in the winter hardiness of barley. The levels of intercellular peroxidase, catalase, and SOD activities on the unit protein basis were 394, 18, and 9% of those of cellular activities, respectively. Major intercellular peroxidase isoforms consisted of four neutrals and four basic forms; whereas major cellular isoforms were two basic forms. Out of the two major catalase isoforms a higher molecular weight form was predominantly abundant in both cellular and intercellular compartments. Among the five major cellular SOD isoforms, three were also present in the intercellular compartment. The presence of substantial amounts of intercellular antioxidant enzymes in overwintering barley leaves may suggest the involvement of these enzymes in the tolerance mechanism to the various stresses that occur during winter.