Indomethacin is a potent, time-dependent, nonselective inhibitor of the cyclooxygenase enzymes (COX-1 and COX-2). Deletion of the 2'-methyl group of indomethacin produces a weak, reversible COX inhibitor, leading us to explore functionality at that position. Here, we report that substitution of the 2'-methyl group of indomethacin with trifluoromethyl produces CF3-indomethacin, a tight-binding inhibitor with kinetic properties similar to those of indomethacin and unexpected COX-2 selectivity (IC50 mCOX-2 = 267 nM; IC50 oCOX-1 > 100 μM). Studies with site-directed mutants reveal that COX-2 selectivity results from insertion of the CF3 group into a small hydrophobic pocket formed by Ala-527, Val-349, Ser-530, and Leu-531 and projection of the methoxy group toward a side pocket bordered by Val-523. CF3-indomethacin inhibited COX-2 activity in human head and neck squamous cell carcinoma cells and exhibited in vivo anti-inflammatory activity in the carrageenan-induced rat paw edema model with similar potency to that of indomethacin.