The importance of normal intracellular glutathione (GSH) levels in the activation of human peripheral blood lymphocytes (PBL) by mitogenic lectins was explored using 2-cyclohexene-1-one (2-CHX-1), an agent that selectively decreases the levels of intracellular GSH. PBL incubated with mitogenic lectins and graded doses of 2-CHX-1 showed a dose-dependent inhibition of activation assayed by [3H]-thymidine uptake and percentage of blast transformation. 2.5 x 10(-5) M 2-CHX-1, a concentration that decreases GSH levels to less than 20% of control, caused an 80 to 90% suppression of both [3H]-thymidine uptake and blast transformation; lower concentrations were less effective. Time course studies showed that 2-CHX-1 was maximally effective only if added during the first 4 hr of culture. At the concentrations used, 2-CHX-1 was not cytotoxic to lectin-stimulated or unstimulated PBL and did not interfere with mitogen-lymphocyte interaction. These results suggest that adequate levels of glutathione are necessary for lymphocyte activation and that the glutathione requirement is exerted in the early activation sequence.