Murine L5178Y lymphoma cells in culture required preformed cyst(e)ine and methionine for growth. Cystathionine was not an adequate substitute for either of these amino acids. 35S-Amino acid uptake and 35S incorporation into cellular reduced glutathione (GSH) occurred rapidly with cysteine or cystine. [ 35S]Methionine uptake was rapid but 35S failed to be incorporated into GSH. The cystathionine pathway for cysteine biosynthesis was absent in this cell line based upon the absence of γ-cystathionase in cell-free extracts and the inability of methionine sulfur to be incorporated into GSH. Glutathione S-transferase activity of intact cells permitted extensive depletion of glutathione with diethylmaleate. Methionine in the medium failed to support the repletion of GSH while cystine from the medium accounted for all of the cysteinyl sulfur in the repleted GSH. Very limited efflux of GSH, and no efflux of oxidized glutathione or disulfide of GSH and cysteine (CYSSG) could be observed in the presence or absence of the irreversible γ-glutamyl transpeptidase inhibitor l-(α S,5 S)-α-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid (AT-125). However, up to 1.9 nmol/10 6 cells of CYSSG accumulated intra-cellularly in cells placed in medium containing 0.5 m m cystine after diethylmaleate depletion of GSH. γ-Glutamyl transpeptidase activity in cell extracts was sufficient to indicate a role for this enzyme in glutathione turnover in L5178Y cells. Under normal culture conditions intracellular glutathione concentration was 2.3 ± 0.7 nmol/10 6 cells.