Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.