Although reactive oxygen species (ROS) have long been suspected to play a key role in Fas (CD95)-induced cell death, the identity of specific ROS involved in this process and the relationship between apoptotic and necrotic cell death induced by Fas are largely unknown. Using electron spin resonance (ESR) spectroscopy, we showed that activation of Fas receptor by its ligand (FasL) in macrophages resulted in a rapid and transient production of hydrogen peroxide (H2O2) and hydroxyl radicals (·OH). The response was visible as early as 5 min and peaked at approximately 45 min post-treatment. Morphological analysis of total death response (apoptosis vs. necrosis) showed dose and time dependency with apoptosis significantly increased at 6 h after the treatment, while necrosis remained at a baseline level. Only at a 35-fold increase in apoptosis did necrosis become significant. Inhibition of apoptosis by a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD-fmk), significantly inhibited cell necrosis, indicating the linkage between the two events. Catalase (H2O2 scavenger) and deferoxamine (·OH scavenger) effectively inhibited the total death response as well as the ESR signals, while superoxide dismutase (SOD) (O scavenger) had minimal effects. These results established the role for H2O2 and ·OH as key participants in Fas-induced cell death and indicated apoptosis as a primary mode of cell death preceding necrosis. Because the Fas death pathway is implicated in various inflammatory and immunologic disorders, utilization of antioxidants and apoptosis inhibitors as potential therapeutic agents may be advantageous. © 2004 Wiley-Liss, Inc.