Reactive oxygen species (ROs) are known to be involved in many physiological and pathological processes. Initially ROs-producing NaDph oxidase (NOX) proteins were thought to be present in phagocytes. however, recent studies have demonstrated that NOX proteins are expressed in many other cell types and tissues. NOX family members' expression and function seems to vary from tissue to tissue. We determined the expression of the NOX family of proteins (NOX1-5) in normal breast tissue and breast tumors. Our study revealed that normal breast tissues express NOX1, 4 and 5 genes. similar pattern of expression was revealed in a breast epithelial cell line. We found that NOX4 was overexpressed in the majority of breast cancer cell lines and primary breast tumors. NOX4 was also overexpressed in ovarian tumors. Overexpression of NOX4 in normal breast epithelial cells resulted in cellular senescence, resistance to apoptosis, and tumorigenic transformation. Overexpression of NOX4 in already transformed breast tumor cells also showed increased tumorigenicity. strong evidence suggests that regulation of these processes occurs through NOX4 generation of ROs in the mitochondria. We demonstrate that the NOX4 protein contains a 73 amino acid long mitochondrial localization signal at the N-terminus that is capable of transporting a passenger protein GFp into the mitochondria. Treatment of NOX4 overexpressing cells with catalase resulted in decreased tumorigenic characteristics. Together, this study provides evidence for an oncogenic function for NOX4 protein localized to mitochondria and suggests that NOX4 is a novel source of ROs produced in the mitochondria. This study also identifies a possible treatment of NOX4-induced breast cancer by antioxidant treatment. © 2010 Landes Bioscience.