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Reactive oxygen species (ROS) have traditionally been viewed as a toxic group of molecules; however, recent publications have shown that these molecules, including H₂O₂, can also strongly promote cell survival. Even though the retina has a large capacity to produce ROS, little is known about its non‐mitochondrial sources of these molecules, in particular the expression and function of NADPH oxidase (Nox) proteins which are involved in the direct generation of superoxide and indirectly H₂O₂. This study demonstrated that 661W cells, a retina‐derived cell line, and mouse retinal explants express Nox2, Nox4 and certain of their well‐established regulators. The roles of Nox2 and Nox4 in producing pro‐survival H₂O₂ were determined using 661W cells and some of the controlling factors were identified. To ascertain if this phenomenon could have physiological relevance, the novel technique of time‐lapse …