Peroxisome proliferator-activated receptor gamma (PPARgamma) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPARgamma. Here, we report that some PPARgamma ligands, including 15-deoxy-Delta12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPARgamma ligand binding pocket through a Michael addition reaction by an alpha,beta-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPARgamma, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the alpha,beta-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the alpha,beta-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPARgamma. We propose that PPARgamma senses oxidation of fatty acids by recognizing such an alpha,beta-unsaturated ketone as a common moiety.