Administration of gamma-glutamylcystine or of gamma-glutamylcysteine disulfide to mice leads to significantly increased levels of glutathione in the kidney as compared to controls given glutamate plus cysteine (or cystinylbisglycine). Studies with gamma-glutamylcystine selectively labeled with 35S in either the internal or external S atom indicate preferential utilization of the gamma-glutamylcysteine moiety of this compound for glutathione synthesis. Mice depleted of glutathione by treatment with buthionine sulfoximine do not significantly use the disulfides gamma-glutamylcystine or gamma-glutamylcysteine disulfide but do use gamma-glutamylcysteine for glutathione synthesis. These findings suggest a pathway in which gamma-glutamylcystine, formed by transpeptidation between glutathione and cystine, is transported and reduced by transhydrogenation with glutathione to cysteine and gamma-glutamylcysteine; the latter is used directly for glutathione synthesis. The findings show transport of gamma-glutamyl amino acids, indicate an alternative pathway of glutathione synthesis, and demonstrate a means of increasing kidney glutathione levels.