To determine Fas-induced reactive oxygen species (ROS) generation in cells sensitive to Fas-mediated apoptosis, chemiluminescence, a strong indicator of ROS generation, was monitored after stimulation with agonistic anti-Fas. Fas ligation resulted in a rapid and sustained ROS generation. The generation reached a maximum within 5 min and was still observed at least 40 min after the stimulation. No significant ROS generation was observed under conditions where apoptosis was absent. Furthermore, an NADPH oxidase inhibitor, diphenylene iodonium, prevented Fas-mediated ROS generation, suggesting that Fas induces ROS generation via stimulation of the oxidase system.