Reperfusion injury, the paradoxical tissue response that occurs after the restoration of blood flow and oxygenation following ischemia, has been a focus of research for over four decades. Excess production of reactive oxygen species (ROS) is a critical factor in this phenomenon, and efforts have been made to identify the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Xanthine oxidase, NADPH oxidase (iNOS), mitochondria, and uncoupled nitric oxide synthase have been identified as the most likely contributors to reperfusion-induced oxidative stress and are priority targets for therapeutic intervention. While all four sources are present in most tissues, specific sources are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The involvement of multiple ROS sources in reperfusion injury is supported by evidence showing that ROS produced by one source can activate and enhance ROS production by another source. This review provides an overview of the evidence implicating ROS in reperfusion injury, its clinical implications, and the current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue.Reperfusion injury, the paradoxical tissue response that occurs after the restoration of blood flow and oxygenation following ischemia, has been a focus of research for over four decades. Excess production of reactive oxygen species (ROS) is a critical factor in this phenomenon, and efforts have been made to identify the dominant cellular and enzymatic sources of excess ROS production following ischemia-reperfusion (I/R). Xanthine oxidase, NADPH oxidase (iNOS), mitochondria, and uncoupled nitric oxide synthase have been identified as the most likely contributors to reperfusion-induced oxidative stress and are priority targets for therapeutic intervention. While all four sources are present in most tissues, specific sources are enriched in certain tissues, such as xanthine oxidase in the gastrointestinal tract and mitochondria in the metabolically active heart and brain. The involvement of multiple ROS sources in reperfusion injury is supported by evidence showing that ROS produced by one source can activate and enhance ROS production by another source. This review provides an overview of the evidence implicating ROS in reperfusion injury, its clinical implications, and the current understanding of the four most frequently invoked enzymatic sources of ROS production in post-ischemic tissue.