Fibroblasts from patients with the disease 5-oxoprolinuria have reduced glutathione synthetase activity and are thus glutathione (GSH) deficient. In this study, 5-oxoprolinuria fibroblasts (GM3877 cells) contained less GSH than normal diploid fibroblasts as determined by biochemical analysis and by flow cytometry using monochlorobimane. They also contained lower gamma-glutamylcysteine synthetase activity than normal cells. However, cocultures of GM3877 cells and normal cells displayed either normal or slightly elevated GSH content, depending upon the assay used. When differentially labeled with fluorescent beads, cocultured, and then isolated by fluorescence-activated cell sorting, both GM3877 cells and normal cells had GSH content similar to that of sorted normal cells cultured alone, whereas sorted GM3877 cells cultured alone showed depressed GSH content. GM3877 cells had detectable levels of gamma-glutamylcysteine (gamma-GC) when cultured alone, but gamma-GC was undetectable in these cells when they were cocultured with normal cells, indicating that it was efficiently metabolized to GSH by the normal cells. These changes in low-molecular-weight thiols were likely to have been mediated by metabolic cooperation across gap junctions because they were dependent upon confluency and because media conditioned by either cell type failed to significantly alter the GSH content of the other cell type. Cocultures exposed to moderate levels of hydrogen peroxide showed less depletion of GSH than GM3877 cells cultured alone, suggesting that the sharing of low-molecular-weight thiols or other reductants via metabolic cooperation can protect cells from oxidative stress.