Reactive oxygen species (ROS) have emerged as intracellular signaling molecules in multiple cellular processes such as proliferation, apoptosis, and senescence. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, particularly protein tyrosine phosphatases (PTPs). Recent studies suggested that the abnormally high levels of cellular ROS production are concurrent with the development of human diseases such as cancers. We examined the role of ROS in the control of signal transduction in Ras- and Src-transformed NIH-3T3 cells which constitutively produce more ROS and possess more active tyrosine phosphorylation signals than parental NIH-3T3 cell. Treatment of transformed cells with diphenyleneiodonium (DPI), a NADPH oxidase (Nox) inhibitor, led to the suppression of cellular ROS production. Under this condition, we found that the activity of endogenous PTPs was elevated, whereas the tyrosine phosphorylation level of cellular proteins was decreased. Such an effect was more pronounced in Ras-transformed cells, compared with that in Src-transformed cells, suggesting that Ras-mediated signal transduction is essentially ROS-dependent. This hypothesis was further investigated. In Ras-transformed cells treated with DPI, a 120 kDa protein, which showed a significant decrease of tyrosine phosphorylation, was subsequently identified as focal adhesion kinase (FAK). Upon the inhibition of cellular ROS production, FAK was dephosphorylated at tyrosine 397 (Tyr397), an integrin-induced auto-phosphorylation site whose phosphorylation activates FAK and enhances cell migration. Treatment of DPI also led to the decrease of phosphorylation levels of Tyr576, Tyr577, Tyr861 and Tyr925 of FAK, concurrent with the decreased kinase activity of Src that recognizes and phosphorylates those Tyr residues in FAK. We also observed that the overexpression of dominant negative Rac1N17, which has been shown to block Nox activity, resulted in the dephosphorylation of FAK and Src. Furthermore, we showed that Ras-transformed cells lost their membrane protrusions in a time-dependent manner in response to DPI treatment, concomitant with the reactivation of cellular PTPs that were originally undergoing reversible oxidation. Our data thus suggested that the Ras-mediated migration signal is regulated by the ROS-dependent activation of FAK and Src through constitutive oxidation and inactivation of endogenous PTPs.