This paper shows the effects of re-aeration on the glutathione pool following a prolonged period of root hypoxia. An increased content of total glutathione has been measured in roots of wheat seedlings (Triticum aestivum L. cv. Alcedo), grown in a nitrogen-flushed nutrient solution (HI) with their shoots in air compared with roots of aerobically grown plants (C). Re-aeration of hypoxically pretreated roots causes oxidative injury indicated by the oxidation of reduced glutathione (GSH), decrease of total thiol-groups and increased formation of TBA reactive material (lipid peroxidation). Re-admission of oxygen results in a 50% rise in oxygen uptake over the whole 16 h re-aeration period compared with the control. During this time the overall glutathione pool of HI treatment increases to almost double that of the control, essentially reflected in the amount of oxidized glutathione (GSSG). Hypoxically pretreated roots showed lower glutathione reductase activity (GR) than the control. Immediately following re-aeration the activity was further decreased to a limiting value which seems to prevent full reduction of the newly formed glutathione. Therefore, the capacity to reduce the GSSG pool is below the capacity for net glutathione synthesis. This results in a decline of the GSH/GSSG ratio which reflects oxidative stress. The enzyme activity recovers slowly after re-aeration exceeding the values of aerobically grown roots only after 16 h correlating with a high reduction state of the glutathione pool. Copper, known to induce the formation of reactive oxygen species, strengthened the effect of re-aeration and enhanced the post-anoxic injury irreversibly. The importance of the glutathione system in roots to cope with varying oxygen tension is discussed.