Reactive oxygen species (ROS) or reactive oxygen intermediates (ROIs) mediate complex signaling involving multiple pathways. In this report, we demonstrate for the first time that endogenous Bruton's tyrosine kinase (Btk) and Akt can interact with each other in DT40 chicken B cells and human Nalm6 B cells and that this interaction is inducible following H2O2 stimulation. This interaction is supported by visualizing the co-localization of Btk and Akt in the perinuclear region and membrane ruffles in COS-7 cells. We have also shown the involvement of phosphatidylinositol 3-kinase (PI 3-K) and Btk in the phosphorylation of Akt following stimulation by hydrogen peroxide (H2O2). Interestingly, Akt phosphorylation was found in the presence of Btk even in the absence of oxidative stress. In addition, we have investigated the involvement of PI 3-K in the MAPKs and ERK and JNK phosphorylation, in the presence or absence of Btk. Phosphorylation of both ERK and JNK increased when the PI 3-K pathway was inhibited and both pathways were modulated positively by Btk. Taken together, based on the study of endogenous conditions, we show the novel interaction of Btk and Akt in H2O2 signaling in B cells. (C) 2002 Elsevier Science (USA). All rights reserved.