Tumor necrosis factor (TNF) is known to mediate its signaling through generation of reactive oxygen species (ROS), but the type of TNF signal regulated by ROS and the nature of the ROS species involved are not fully understood. In this report, we investigated the effect of various superoxide radical quenchers--pyrrolidine dithiocarbamate (PDTC), N-acetyl-L-cysteine (NAC), and glutathione (GSH)--an hydroxyl radical quencher (mannitol), and lipid peroxide quenchers--butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)--on TNF-induced activation of nuclear transcription factors-kappa B (NF-kappa B) and activator protein-1 (AP-1), c-jun amino-terminal kinase (JNK), and apoptosis in human monocytic U937 cells. TNF-induced NF-kappa B activation was inhibited by both superoxide and lipid peroxide quenchers but potentiated by an hydroxyl radical quencher. In contrast, none of the radical quenchers had any significant effect on TNF-induced AP-1 activation. TNF-induced JNK activation, similar to NF-kappa B, was inhibited by both superoxide and lipid peroxide quenchers but potentiated by hydroxyl radical quencher. TNF-induced activation of caspase activity was blocked by all three types of quenchers. TNF cytotoxicity, however, was potentiated by superoxide radical quenchers and suppressed by hydroxyl radical and lipid peroxide quenchers. Overall, these results suggest that hydroxyl radicals mediate TNF-induced apoptosis but not activation of NF-kappa B, AP-1, and JNK; superoxide radicals mediate NF-kappa B and JNK activation but potentiate apoptosis; and lipid peroxides are required for all the signals induced by TNF.