Oxidative stress is implicated in human diseases. Some of the oxidative pathways are harbored in the mitochondria. NAD(P)H oxidases have been identified not only in phagocytic but also in somatic cells. Nox4 is the most ubiquitous of these oxidases and is a major source of reactive oxygen species (ROS) in many cell types and in kidney tissue of diabetic animals. We generated specific Nox4 antibodies, and found that Nox4 localizes to mitochondria. (i) Immunoblot analysis in cultured mesangial cells and kidney cortex revealed that Nox4 is present in crude mitochondria, in mitochondria-enriched heavy fractions, and in purified mitochondria; (ii) immunofluorescence confocal microscopy also revealed that Nox4 localizes with the mitochondrial marker Mitotracker; and (iii) the mitochondrial localization prediction program MitoProt indicated that the probability score for Nox4 is identical to mitochondrial protein cytochrome c oxidase subunit IV. We also show that in purified mitochondria, siRNA-mediated knockdown of Nox4 significantly reduces NADPH oxidase activity in pure mitochondria and blocks glucose-induced mitochondrial superoxide generation. In a rat model of diabetes, mitochondrial Nox4 expression is increased in kidney cortex. Our data provide evidence that a functional Nox4 is present and regulated in mitochondria, indicating the existence of a previously undescribed source of ROS in this organelle.