The effect of osmotic stress on glutathione and hydroxymethylglutathione levels was compared in three wheat genotypes and two 5A chromosome substitution lines. Freezing-tolerant genotypes seemed also to be tolerant to osmotic stress induced by polyethylene glycol (PEG), since their fresh weight was not affected by the treatment. However, the growth of freezing-sensitive genotypes was reduced by 7-day PEG treatment and they had greater injuries after osmotic stress. The reduced forms of the two glutathione precursors, cysteine and gamma-glutamylcysteine, and of hydroxymethylglutathione (hmGSH) and glutathione (GSH) were present in greater quantities after PEG treatment in the two tolerant genotypes than in the sensitive ones. Similarly, osmotic stress resulted in a higher ratio of the reduced to the oxidised form of these thiols and in greater activity of gamma-glutamylcysteine synthetase and glutathione reductase in the tolerant genotypes compared to the sensitive ones. Following in vivo glutathione synthesis, a greater incorporation of radioactivity from [35S]sulphate into the four thiols was found in the tolerant genotypes than in the sensitive ones during osmotic stress. The present results indicate that hmGSH and GSH may contribute to the improvement of tolerance against osmotic stress in wheat and that the 5A chromosome influences the stress-induced changes in GSH and hmGSH levels.