A significant part of the therapeutic benefit of interferon-alpha (IFN-alpha) therapy in malignant diseases and in chronic viral infections is assumed to result from activation of lymphocytes with natural killer (NK) and T cell phenotype. In tumor tissue and in chronically infected tissue, the function and viability of these lymphocytes are frequently impaired. Mononuclear phagocyte (MP)-derived reactive oxygen species (ROS) have been proposed to contribute to the lymphocyte suppression in these tissues. Here, we report that three types of human cytotoxic lymphocytes of relevance to immunoactivation by IFN-alpha, CD3epsilon+/8+/56- T cells, CD3epsilon-/56+ NK cells, and CD3epsilon+/56+ NK/T cells became anergic to IFN-alpha induction of the cell-surface activation marker CD69 after exposure to autologous MPs in vitro. In addition to their incapacity to express CD69, cytotoxic lymphocytes acquired features characteristic of apoptosis after incubation with MPs. The lymphocyte apoptosis and nonresponsiveness to IFN-alpha were prevented by two inhibitors of reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent formation of ROS in MPs, histamine dihydrochloride and diphenylene ionodonium, as well as by catalase, a scavenger of ROS. We conclude that MP-derived ROS may negatively affect IFN-alpha-induced immunostimulation and propose that ROS inhibitors or scavengers may be useful to improve lymphocyte activation during treatment with IFN-alpha.