Objectives: Cyclopentenone prostaglandins, such as delta12-prostaglandin J2 (d12-PGJ2), have been shown to promote bone formation in vitro and in vivo. These compounds are also the natural ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) subtype of nuclear hormone receptor superfamily. However, the mechanisms by which d12-PGJ2 mediates its bone formation effect through the PPAR-gamma receptor are unclear. The purpose of this study was to investigate whether PPAR-gamma mediates the effect of d12-PGJ2 on bone nodule formation in vitro. Methods: MC3T3-E1 mouse osteoblast-like cells were incubated in media containing 0 (control), 1, 3, and 10µM of d12-PGJ2 simultaneously with 0, 0.1, and 1µM of irreversible PPAR-gamma antagonist, GW 9662, either in the first half (days 0-9) or in the second half (days10-18) of total incubation period (18 days). Cells were stained with 1% alizarin red in 1% ammonium hydroxide to identify calcium precipitation. The percentage of area of bone nodule formation was examined using image analysis program (Scion Image). Results: d12-PGJ2 at 3µM significantly increased area of nodule formation (138% increase, p<0.05) during the first half of incubation but addition of GW 9662 had no effect on d12-PGJ2-induced nodule formation. In contrast, only 45% increase in nodule formation was observed at 10 mM d12-PGJ2 during the second half of incubation. Moreover, GW 9662 at 0.1 and 1mM inhibited d12-PGJ2-induced nodule formation (66% inhibition, p<0.05) during this incubation period. Conclusions: These findings suggest that d12-PGJ2-induced bone formation is mainly occurred in the early stage of nodule formation and mediated by PPAR-gamma independent mechanism. In contrast, PPAR-gamma dependent mechanism is a possible pathway in d12-PGJ2-induced bone formation during the late stage of nodule formation. This work was supported by DE-PO1-08917 and Orapharma, Inc.