Hydrogen peroxide, a reactive oxygen intermediate produced by activated neutrophils, has been shown to inhibit the response of human T lymphocytes to mitogens and alloantigens. Since hydrogen peroxide is known to react with iron and to induce lipid peroxidation, we compared the effects of hydrogen peroxide and a lipid peroxidation product, malondialdehyde, on the response of human peripheral blood mononuclear cells to T-cell mitogens. Peripheral blood mononuclear cells pretreated with 1 mmol/L of malondialdehyde, washed, and resuspended in fresh medium exhibited no inhibition of phytohemagglutinin responsiveness. Peripheral blood mononuclear cells treated in the same manner but with 200 mumol/L of hydrogen peroxide were inhibited by more than 95%. The addition of ferric edetate did not alter the inhibitory effects of 50 to 100 mumol/L of hydrogen peroxide, nor did the addition of deferoxamine, an iron chelator. These studies suggest that exogenous lipid peroxidation does not affect lymphocyte activation but that hydrogen peroxide has a direct inhibitory effect. Although monocytes are necessary for T-cell mitogenic responses, the effect of hydrogen peroxide was found to be directed at T lymphocytes. Exposure of T cells to a single dose of 200 mumol/L of hydrogen peroxide resulted in more than 71% suppression of the proliferative response measured 48 hours later, but the effect was spontaneously reversed by 72 to 96 hours. Repeated exposure of the cells to hydrogen peroxide resulted in continued inhibition of the proliferative response. These findings suggest that hydrogen peroxide produced by inflammatory phagocytic cells might be capable of suppressing the immune response of nearby T lymphocytes.