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Turnover of cellular glulathione in isolated rat kidney cells was studied using cystine or methionine as sulfur donor. In the absence of any sulfur donor a continuous decrease of intracellular reduced glutathione (GSH) during incubation of the cells was observed. This decrease was abolished in the presence of cystine and, as indicated by incorporation of 35S, there was also a rapid synthesis of GSH. In the presence of a γ-glutamyltransferase inhibitor, the synthesis of intracellular GSH was accompanied by an accumulation of extracellular cysteine-glutathione mixed disulfide whereas only minor amounts of GSH and glutathione disulfide could be detected. The intracellular levels of both the cysteine-glutathione and glutathione disulfides were at all time points very low. Even though the uptake of cysteine was rapid and not rate-limiting for GSH synthesis, almost no cystine could be detected intracellularly. An increasing intracellular cysteine concentration was however observed, indicating a paid reduction of cysteine. In contrast of cysteine, methionine did not protect from the loss of intracellular GSH and only a low rate of incorporation of 35S into GSH was observed. Methionine was rapidly taken up into the cells but was apparently converted to cysteine only a very limited extent. This is most likely due to a low activity of the enzyme cystathionase since neither homocystine nor cystathionine was very effective in supporting GSH synthesis. [ABSTRACT FROM AUTHOR]