It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that beta Pix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and beta Pix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of beta Pix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of beta Pix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, beta Pix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation. (c) 2005 Elsevier Inc. All rights reserved.