In order to explore the role of glutathione in cell-mediated cytotoxicity, we have examined the effect of the sulphydryl-reactive and glutathione-depleting agent 2-cyclohexene-1-one on antibody-dependent cellular cytotoxicity, spontaneous cell-mediated cytotoxicity, and cell-mediated lympholysis by human peripheral blood mononuclear cells. 2-Cyclohexene-1-one significantly inhibited (P less than 0.001) both antibody-dependent and spontaneous cell-mediated cytotoxicity using three different cell-line targets, at three different killer:target cell ratios (10:1, 25:1 and 50:1). Using K-562 cell-line targets, spontaneous cell-mediated cytotoxicity was inhibited by 2-cyclohexene-1-one with an ID50 of 0.71 X 10(-4) M-1.48 X 10(-4) M, while antibody-dependent cellular cytotoxicity was less sensitive to inhibition, and required slightly higher concentrations of 1.48 X 10(-4) M-3.98 X 10(-4) M to achieve 50% inhibition. Similar results were seen with human colon tumour cell-line and Chang liver cell-line cells as targets. Maximal inhibition occurred when 2-cyclohexene-1-one was added to the cytotoxicity assay 60 min prior to, at the start of, or within the first 60 min of a 4-hr assay; inhibition of cytotoxicity occurred with pretreatment of effector cells; and no inhibition of cytotoxicity was observed with pretreatment of target cells. Both the allogeneic mixed leucocyte reaction and cell-mediated lympholysis were also significantly inhibited (P less than 0.001) by 2-cyclohexene-1-one. These studies demonstrate that 2-cyclohexene-1-one is an effective inhibitor of cell-mediated cytotoxicity and suggest that glutathione, specific glutathione-protein interactions, or protein-bound sulphydryl groups are involved in allowing cells to carry out cytolysis.