The dependence of intracellular glutathione, an important radical scavenger, on the extracellular glutamate and cystine concentration and the velocity of the high affinity sodium/glutamate transporter was studied in freshly-isolated Müller glial cells of the guinea-pig, kept in vitro for up to 11h. To this end the relative Müller cell glutathione levels were measured using the fluorescent dye monochlorobimane, using different concentrations of glutamate and cystine in Ringer solution. In some experiments l-buthionine-[S,R]-sulfoximine, a blocker of glutathione synthesis, or l-trans-pyrrolidine-2,4-dicarboxylic acid and l-α-aminoadipic acid, inhibitors of glutamate uptake, were added. The Müller cells maintained about 80% of the normal glutathione level when maintained in Ringer solution containing 100μM glutamate for 11h. When under these conditions 100μM cystine was added, the glutathione level increased to values, which were even higher than those at the beginning of the incubation period. Addition of cystine without glutamate caused a run down of the glutathione level to about 45% of the normal level, which is comparable to the run down in pure Ringer solution. Likewise, application of l-buthionine-[S,R]-sulfoximine (5mM) lead to a strong run down of the glutathione level even in glutamate/cystine (100μM)- containing solution. A similar suppressing effect was observed using l-trans-pyrrolidine-2,4-dicarboxylic acid and l-α-aminoadipic acid in the presence of 100μM cystine and glutamate. We conclude that the intracellular glutamate concentration of the Müller cells is determined by the extracellular glutamate concentration and the velocity of the sodium/glutamate uptake. Consequently, cystine uptake into Müller cells, which is performed by the cystine/glutamate antiporter, is fuelled by the sodium/glutamate transporter with intracellular glutamate. Both glutamate and cystine are also substrates for glutathione synthesis. The glutathione level is logically limited by the capacity of the sodium/glutamate transporter to provide glutamate intracellularly for, first, cystine uptake and, second, direct insertion into glutathione. Accordingly, the glutathione level is reduced when the sodium/glutamate transporter is blocked. Thus, a diminution of the glutathione level should be taken into consideration when the effects of sodium/glutamate uptake failure and reduced intracellular glutamate concentrations are discussed.