A number of agents capable of interfering with oxidative events were found to inhibit, in a dose-dependent manner, DNA synthesis in isolated human peripheral blood lymphocytes stimulated with phytohaemagglutinin, or phorbol myristate acetate plus ionomycin. These inhibitory substances were: the iron chelators desferrioxamine and desferrithiocin; the electron acceptor ferricyanide; the anti-oxidant nordihydroguaiaretic acid; ebselen, an agent with glutathione peroxidase-like activity; and diphenylene iodonium, an inhibitor of NADPH-oxidase. The actions of desferrioxamine and desferrithiocin were abolished by prior saturation with iron. Ferrocyanide was much less active in inhibiting human lymphocyte DNA synthesis than its redox partner ferricyanide. Desferrioxamine, ferricyanide and nordihydroguaiaretic acid also inhibited lipopolysaccharide-initiated DNA synthesis in mouse splenocytes in vitro. The common property of these structurally dissimilar agents is their ability to prevent formation of, or detoxify, reactive oxygen species. Thus, the data are consistent with an obligatory role for reactive oxygen formation in human T-cell and mouse B-cell activation at a stage prior to DNA synthesis.