The production of reactive oxygen species (ROS) accompanies many signaling events. Antioxidants and ROS scavenging enzymes in general have effects that indicate a critical role for ROS in downstream signaling, but a mechanistic understanding of the contribution of ROS as second messengers is incomplete. Here, the role of reactive oxygen species in cell signaling is discussed, emphasizing the ability of ROS to directly modify signaling proteins through thiol oxidation. Examples are provided of protein thiol modifications that control signal transduction effectors that include protein kinases, phosphatases, and transcription factors. Whereas the effects of cysteine oxidation on these proteins in experimental systems is clear, it has proven more difficult to demonstrate these modifications in response to physiologic stimuli. Improved detection methods for analysis of thiol modification will be essential to define these regulatory mechanisms. Bridging these two areas of research could reveal new regulatory mechanisms in signaling pathways, and identify new therapeutic targets.