Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated. No effects were observed on the intrinsic activities of H-Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.