13C-NMR spectroscopy was employed non-invasively for the real-time measurement of the rates of glutathione synthesis in intact rabbit lenses supported in organ culture containing L-[3-13C]cysteine. Supplementation of the organ culture medium with 2-mercaptoethanol resulted in a dose- dependent enhancement of lenticular glutathione synthesis rates (dose for 50% maximal effect = 125 microM). At the most effective concentration (400 microM) 2-mercaptoethanol increased the rate of glutathione synthesis 163% relative to the rate observed under control conditions. The mechanism of action for this effect was investigated in intact lenses using antagonists of specific amino acid uptake systems. These experiments demonstrated that the enhanced rates of glutathione synthesis observed in the presence of 2-mercaptoethanol were due to the affinity of the mixed-disulfide formed between cysteine and 2-mercaptoethanol for L system amino acid uptake, thereby providing a mechanism for increasing intracellular cysteine levels by circumventing normal cellular cysteine uptake pathways in the lens. Because of the role of cysteine as the rate limiting substrate in lenticular glutathione biosynthesis, these results suggest a potential strategy to prevent tissue opacification associated with depleted glutathione levels.