Glutathione (GSH) has been implicated in lymphocyte activation and differentiation, as well as in protection from radiation damage. Since [³H]thymidine ([³H]TdR) at high concentrations in the nucleus causes radiation damage to the cells, it is important to rule out the possibility that changes in [³H]TdR uptake by mitogen-activated lymphocytes are not caused by ³H-induced cell injury following alterations in intracellular GSH concentration. In this study, flow-cytometric analysis of cell cycle was used to measure lymphocyte activation. Intracellular GSH levels were enhanced using 2-l-oxothiazolidine-4-carboxylate (OTC) and 2-mercaptoethanol (2ME), which deliver cysteine intracellularly, and suppressed by buthionine sulfoximine (BSO) which inhibits γ-glutamylcysteine synthetase. Enhancement of intracellular GSH concentrations in lymphocytes with 2-oxothiazolidine-4-carboxylate or 2-mercaptoethanol augments mitogen-induced lymphocyte activation, and proliferation, while suppression of intracellular GSH levels by buthionine sulfoximine inhibits the progression of cellular proliferation—but not activation, as measured by flow cytometry. There was a linear relationship between intracellular GSH concentration and conA-activated cells by flow cytometry and between GSH concentration and [³H]TdR incorporation as measured at 24 h. We conclude that alterations of intracellular GSH concentrations may be one way to modulate lymphocyte activation and differentiation.