Reperfusion injury, the paradoxical tissue response following the restoration of blood flow after ischemia, has been a focus of research for over 40 years. Excess production of reactive oxygen species (ROS) is a critical factor in reperfusion injury, with xanthine oxidase, NADPH oxidase, mitochondria, and uncoupled nitric oxide synthase being the most likely contributors. These enzymes are prioritized for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. While all four enzymatic sources are present in most tissues, specific sources like xanthine oxidase in the gastrointestinal tract and mitochondria in the heart and brain are emphasized. Evidence shows that multiple ROS sources contribute to reperfusion injury in most tissues, with redox-signaling enabling ROS from one source to enhance production by another. This review summarizes 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. The concept of reperfusion injury has evolved over the past 30 years, with extensive research on the enzymatic and cellular sources of ROS, the magnitude of ROS production, and how ROS lead to tissue injury. The review addresses the evolution of the concept of ROS-mediated reperfusion injury and examines the evidence supporting or refuting its existence in different organ systems and pathological states. Reperfusion injury can jeopardize the functional recovery of patients with transient blood perfusion disruption, and conditions like sickle cell disease, osteoarthritis, and Alzheimer's disease are now being considered as clinical manifestations of I/R injury. ROS contribute to reperfusion injury through various mechanisms, including the generation of superoxide and hydrogen peroxide, and their role in oxidative stress and redox imbalance. The involvement of ROS in I/R injury is supported by studies showing that interventions enhancing ROS scavenging protect against injury, and that ROS generation recapitulates the injury response. The sources of ROS in post-ischemic tissue include xanthine oxidase, NADPH oxidase, the mitochondrial electron transport chain, and uncoupled nitric oxide synthase. Xanthine oxidase is a major source of ROS following I/R, with its activity being influenced by ischemia and reperfusion. NADPH oxidase is also implicated in ROS production, with studies showing that its inhibition reduces I/R injury. The role of ROS in reperfusion injury is supported by evidence from in vitro and in vivo studies, including the use of inhibitors and genetic models. The review highlights the importance of ROS in reperfusion injury and the need for further research to understand the relative importance of reperfusion-dependent vs reperfusion-independent mechanisms in the overall morbidity and mortality of these conditions.Reperfusion injury, the paradoxical tissue response following the restoration of blood flow after ischemia, has been a focus of research for over 40 years. Excess production of reactive oxygen species (ROS) is a critical factor in reperfusion injury, with xanthine oxidase, NADPH oxidase, mitochondria, and uncoupled nitric oxide synthase being the most likely contributors. These enzymes are prioritized for therapeutic intervention against reperfusion-induced organ dysfunction and tissue damage. While all four enzymatic sources are present in most tissues, specific sources like xanthine oxidase in the gastrointestinal tract and mitochondria in the heart and brain are emphasized. Evidence shows that multiple ROS sources contribute to reperfusion injury in most tissues, with redox-signaling enabling ROS from one source to enhance production by another. This review summarizes 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. The concept of reperfusion injury has evolved over the past 30 years, with extensive research on the enzymatic and cellular sources of ROS, the magnitude of ROS production, and how ROS lead to tissue injury. The review addresses the evolution of the concept of ROS-mediated reperfusion injury and examines the evidence supporting or refuting its existence in different organ systems and pathological states. Reperfusion injury can jeopardize the functional recovery of patients with transient blood perfusion disruption, and conditions like sickle cell disease, osteoarthritis, and Alzheimer's disease are now being considered as clinical manifestations of I/R injury. ROS contribute to reperfusion injury through various mechanisms, including the generation of superoxide and hydrogen peroxide, and their role in oxidative stress and redox imbalance. The involvement of ROS in I/R injury is supported by studies showing that interventions enhancing ROS scavenging protect against injury, and that ROS generation recapitulates the injury response. The sources of ROS in post-ischemic tissue include xanthine oxidase, NADPH oxidase, the mitochondrial electron transport chain, and uncoupled nitric oxide synthase. Xanthine oxidase is a major source of ROS following I/R, with its activity being influenced by ischemia and reperfusion. NADPH oxidase is also implicated in ROS production, with studies showing that its inhibition reduces I/R injury. The role of ROS in reperfusion injury is supported by evidence from in vitro and in vivo studies, including the use of inhibitors and genetic models. The review highlights the importance of ROS in reperfusion injury and the need for further research to understand the relative importance of reperfusion-dependent vs reperfusion-independent mechanisms in the overall morbidity and mortality of these conditions.