During flooding, when roots are submerged in oxygen-free water, root tissue becomes hypoxic and its metabolism is characterized by fermentation processes and limited respiratory activity. After returning to aerobic conditions (post-hypoxic period) high respiration rates together with symptoms of oxidative stress are observed. Plant mitochondria have two terminal oxidases: a cytochrome oxidase with high affinity to oxygen and an alternative (cyanide resistant) oxidase (AOX) with a relatively low oxygen affinity. We compared mitochondrial respiration and AOX expression immediately after hypoxic and during post-hypoxic period. Four-day-old barley (Hordeum vulgare L. cv Gregor) seedlings were transferred for 5 days to Knop nutrient medium flushed with air (control) or nitrogen (hypoxia) and after returned for 24 h to aerated nutrient medium (post-hypoxia). NADH/NAD1 +NADH and UQH2/UQtotal ratios increased in hypoxia-treated roots. After the hypoxic roots were returned to aerated medium, increases in respiration rate and ATP concentration were observed. Mitochondria isolated from barley roots at the end of hypoxic period exhibited high respiratory rates as compared to mitochondria from control and post-hypoxic roots. Control root mitochondria expressed high AOX capacity in the presence of pyruvate and DTT. Mitochondria isolated at the end of the hypoxic period were highly cyanide sensitive under AOX- activating conditions, however, after 24 h of post-hypoxia, AOX capacity was comparable to that observed in the control. The capacity of AOX was correlated with the amount of AOX protein determined by Western blotting. Faint or no bands were observed immediately after hypoxia with the AOX protein appearing again during the post-hypoxic period. Examination of AOX transcript levels showed no significant differences between control, hypoxic and post-hypoxic roots indicating that the regulation of AOX expression by oxygen availability in barley roots may take place at the translation level.