The generation of reactive oxygen species (ROS) in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the Rac protein. We report here that the COOH-terminal region of Nox1, a protein related to gp91phox (Nox2) of phagocytic cells, is constitutively associated with βPix, a guanine nucleotide exchange factor for Rac. Both growth factor-induced ROS production and Rac1 activation were completely blocked in cells depleted of βPix by RNA interference. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Moreover, the depletion of Nox1 by RNA interference inhibited growth factor-induced ROS generation. These results suggest that ROS production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, βPix, and Rac1, which then binds to Nox1 to stimulate its NADPH oxidase activity.