The mechanism of the anticolorectal cancer activity of the nonsteroidal anti-inflammatory drug indomethacin is poorly understood. Indomethacin inhibits both cyclooxygenase (COX) isoforms, but it may also act via COX-independent targets. Indomethacin can bind and activate the transcription factor peroxisome proliferator-activated receptor (PPAR) gamma. Moreover, natural and synthetic PPARgamma ligands can induce growth arrest and apoptosis of human colorectal cancer cells in vitro. Therefore, we tested the hypothesis that the antiproliferative activity of indomethacin on human colorectal cancer cells in vitro is explained by a PPARgamma-dependent mechanism of action. Human colorectal cancer cell lines SW480 and HCT116 both expressed functional PPARgamma. Indomethacin directly activated PPARgamma in both cell lines (HCT116 > SW480). A dominant-negative PPARgamma strategy was used to demonstrate that endogenous PPARgamma represses proliferation of HCT116 cells (compatible with tumor suppressor activity) but that the presence of functional PPARgamma is not necessary for the antiproliferative activity (or reduction in cyclin D1 protein) associated with indomethacin in vitro. In summary, indomethacin (>100 microM) directly activates PPARgamma in human colorectal cancer cells. However, PPARgamma activation does not underlie the antineoplastic activity of indomethacin on human colorectal cancer cells in vitro.