In the course of a high throughput screen to search for ligands of peroxisome proliferator activated receptor-γ (PPARγ), we identified GW9662 using a competition binding assay against the human ligand binding domain. GW9662 had nanomolar IC₅₀ versus PPARγ and was 10- and 600-fold less potent in binding experiments using PPARα and PPARδ, respectively. Pretreatment of all three PPARs with GW9662 resulted in the irreversible loss of ligand binding as assessed by scintillation proximity assay. Incubation of PPAR with GW9662 resulted in a change in the absorbance spectra of the receptors consistent with covalent modification. Mass spectrometric analysis of the PPARγ ligand binding domain treated with GW9662 established Cys²⁸⁵ as the site of covalent modification. This cysteine is conserved among all three PPARs. In cell-based reporter assays, GW9662 was a potent and selective antagonist of full-length PPARγ. The functional activity of GW9662 as an antagonist of PPARγ was confirmed in an assay of adipocyte differentiation. GW9662 showed essentially no effect on transcription when tested using both full-length PPARδ and PPARα. Time-resolved fluorescence assays of ligand-modulated receptor heterodimerization, coactivator binding, and corepressor binding were consistent with the effects observed in the reporter gene assays. Control activators increased PPAR:RXR heterodimer formation and coactivator binding to both PPARγ and PPARδ. Corepressor binding was decreased. In the case of PPARα, GW9662 treatment did not significantly increase heterodimerization and coactivator binding or decrease corepressor binding. The experimental data indicate that GW9662 modification of each of the three PPARs results in different functional consequences. The selective and irreversible nature of GW9662 treatment, and the observation that activity is maintained in cell culture experiments, suggests that this compound may be a useful tool for elucidation of the role of PPARγ in biological processes.