Transformed poplars overexpressing γ-glutamylcyst-eine synthetase (γ-ECS) in the chloroplast (Lggs) were used to investigate chloroplastic biosynthesis of glutathione (GSH). In Lggs leaves, GSH contents were enhanced by up to 3.7-fold. In general, the highest GSH contents were observed in lines with highest γ-glutamylcysteine (γ-EC) contents. These lines had relatively low glycine. In darkness, foliar GSH decreased and γ-EC increased. Illumination of pre-darkened Lggs in air resulted in a 5-fold decrease in the γ-EC:GSH ratio. This light-induced decrease was largely abolished if leaves were illuminated at high CO2. Consequently, the γ-EC:GSH ratio of illuminated leaves was much higher at high CO2 than in air. At high CO2 total foliar amino acids were higher, but glycine and serine were lower, than in air. These results suggest that photorespiratory glycine is used in chloroplastic GSH synthesis. Despite this, net CO2 fixation was similar in Lggs to untransformed poplars. Pre-illuminated leaf discs from Lggs, and poplars overexpressing γ-ECS in the cytosol (ggs), were incubated in darkness with a range of metabolites. After 15 h, discs from both types of transformant incubated on water had accumulated high levels of γ-EC and showed marked increases in the γ-EC:GSH ratio. Feeding glycine, serine, glycollate or phosphoserine, attenuated the dark-induced changes in the γ-EC5GSH ratio, whereas 3-phosphoglycerate (PGA), phosphoenolpyruvate, glycerate, and hydroxypyruvate did not. Glycine produced from glycollate was therefore required for maximal GSH accumulation in both the chloroplastic and cytosolic compartment. Production of glycine from PGA failed to meet the demand of increased GSH synthetic capacity.