It has become common in the literature for the presence of an activating phosphorylation on a signaling kinase to be interpreted as an ‘active’ kinase pathway. This unfortunate simplification ignores more subtle means of signal regulation. Many signaling events, including UV irradiation, inflammatory cytokines, and mitogen stimulation, are accompanied by generation of reactive oxygen species. These oxidative species include those that acquire one electron from NADH and those that acquire two electrons from NADH. One-electron oxidoreduction reactions result in the release of high-energy species generated by the unused electron from NADH. These species include superoxide, hydroxyl radical, and others, that likely contribute to damaging modifications of proteins and lipids. Twoelectron oxidoreduction mainly results in the in situ generation of hydrogen peroxide, that can be used by endogenous enzymes to oxidize cysteine residues on proteins. These modifications take several forms, including oxidation to sulfenic acid or formation of disulfides involving intra- and intermolecular connections between proteins or S-thiolation of proteins by glutathione and cysteine. Reversible oxidation of proteins on cysteines is potentially a means for controlling signal transduction, since it adds an often-charged, bulky moiety to the protein primary structure, and is reversible, much like protein modifications resulting from phosphorylation. For example, the activity of the MEKK1 protein kinase is regulated by reversible glutathionylation of a single cysteine residue in the ATP binding pocket. Several other kinases have been shown to be activated or inhibited by reversible cysteine oxidation, though mainly with non-physiologic stimuli. Compared to the study of protein phosphorylation, study of specific oxidation on proteins is difficult. Antibodies against Sglutathionylation have been described, and protein sulfhydryls have been labeled with fluorescent maleimides or iodoacetamides. We have developed an unbiased method of quantifying reversible cysteine oxidation in proteins using an affinity capture technique we call Purification of Reversibly Oxidized Proteins. We have applied this technique to study the regulatory oxidation of the p38 MAPK signaling kinase. We have found that p38 is oxidized following exposure of cells to exogenous hydrogen AG-013736 peroxide or prostaglandin J2, and that oxidation results in kinase inactivation despite continued phosphorylation on the activating residues detected by immunoblot. Prostaglandin J2 is an inflammatory mediator that hasbeen characterized forits roleininducing oxidative signals. As such, the oxidation of p38 following exposure of cells to PGJ2 reflects a relevant physiologic signaling event that might occur during inflammation.