The activation of peroxisome-proliferator-activated receptor-γ (PPARγ), which plays a central role in adipocyte differentiation, depends on ligand-dependent co-activator recruitment. In this study, we developed a novel method of PPARγ ligand screening by measuring the increase in fluorescent polarization accompanied by the interaction of a fluorescent co-activator and PPARγ. Sterol receptor co-activator-1 (SRC-1), a major PPARγ co-activator, was probed by fluorescent TAMRA by the Amber codon fluorescence probe method. Polarization was increased by adding PPARγ ligands to a solution containing labeled SRC-1 (designated TAMRA-SRC-S) and PPARγ. The disassociation constants (Kd) of the PPARγ synthesized ligands, pioglitazone (221 nM), troglitazone (83.0 nM), and 15-deoxy-Δ12,14-prostaglandin J(2) (15d-ΔPGJ(2)) (156 nM), were determined by this method. Farnesol (2.89 µM) and bixin (21.1 µM), which we have reported to be PPARγ ligands, increased the fluorescent polarization. Their Kd values were in agreement with the ED(50) values obtained in the luciferase assay. The results indicate that the method is valuable for screening natural PPARγ ligands.