Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events^,,,. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury^, and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS^,; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the βγ-subunit of G protein (Gβγ) attenuates hydrogen peroxide (H₂O₂)-induced ERK activation in rat neonatal cardiomyocytes. The Gβγ-responsive ERK activation induced by H₂O₂ is independent of ligands binding to G_i-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H₂O₂ increases [³⁵S]GTP-γS binding to cardiac membranes and directly activates purified heterotrimeric G_i and G_o but not G_s. Analysis using heterotrimeric G_o and its individual subunits indicates that H₂O₂ modifies Gα_o but not Gβγ, which leads to subunit dissociation. We conclude that Gα_i and Gα_o are critical targets of oxidative stress for activation of ERK.