Localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion of the same components. It has been suggested that ROS-generating complexes follow the principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model of redox signaling compartmentalization. Nevertheless, evidence of local ROS production and the restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH oxidase isoforms, their adapter proteins, and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant systems which prevent uncontrolled ROS diffusion from sites of generation to the adjacent subcellular compartments; the current data on the specific localization of NADPH oxidase activity and its influence on intracellular processes; and the recent evidence of the ROS diffusion restriction. © 2013 Pleiades Publishing, Ltd.