Aim: To characterize the in vitro bioactivities of rhodanine derivatives as novel peroxisome proliferator-activated receptor (PPAR) γ modulators, based on a hit (SH00012671) identified during high-throughput screening (HTS) of a diverse synthetic compound library, and to preliminarily elucidate the structure-activity elationship of this class of PPARγ agonists. Methods: Full-length PPARγ and retinoid X receptor α (RXRα), biotinylated PPAR response element (PPRE), [3H]BRL49653 (rosiglitazone), and streptavidin-coated FlashPlate or microbeads were used to measure the receptor-binding properties of various compounds based on the scintillation proximity assay (SPA) technology. A recombinant PPRE vec- tor was transiently cotransfected with PPARγ and RXRα plasmids into the African green monkey kidney (CV-1) cells, and the effects of BRL49653 and test com- pounds on transcription mediated by PPARγ were determined by examining lu- ciferase (reporter) responses. 3T3-L1 cells were employed to determine whether the compounds facilitated adipogenesis upon PPARγ activation. Results: Of the 16 000 samples screened with the SPA method, only 1 compound (SH00012671) displayed a similar binding affinity (Ki=186.7 nmol/L) to PPARy as BRL49653, but it was inactive in the cell-based assays. A series of rhodanine derivatives were synthesized based on the core structure of SH00012671 and 8 of them showed agonist activities in both cotransfection and pre-adipocyte differentiation assays. To reduce intrinsic cytotoxicities, the sulphur on the rhodanine was changed to oxygen. This alteration led to a decrease in receptor-binding affinities while modi- fied analogues generally maintained agonist efficacies in the cell-based assays. Of the analogues studied, compound 31 exhibited about 70% the efficacy exerted by BRL49653 in both cotransfection and pre-adipocyte differentiation assays. Conclusion: Through minor chemical modifications on the core structure of the initial HTS hit, SH00012671 was transformed to possess both molecular (PPARγ binding) and cellular (adipogenesis) activities. The rhodanine derivatives re- ported here may represent a new scaffold in further understanding the molecular mechanism of agonism at PPARγ.