Indomethacin is a widely used nonsteroidal anti-inflammatory drug. We studied the human cytochrome P450 (CYP) isoform responsible for indomethacin O-demethylation, the major metabolic pathway for indomethacin. For indomethacin O-demethylase activities, the KM value was 34.6 +/- 5.4 muM and the Vmax value was 14.1 +/- 3.9 pmol/mg/min in human liver microsomes (N = 4). Indomethacin O-demethylase activity in human liver microsomes was competitively inhibited by sulfaphenazole, (S)-warfarin, and tolbutamide and was not affected by alpha-naphthoflavone, (S)-mephenytoin, or erythromycin. Indomethacin O-demethylase activities in microsomes from nine human livers were significantly correlated with tolbutamide hydroxylase activities (r = 0.750, p < 0.05) and not with (S)-mephenytoin 4'-hydroxylase activities. When the capacity for indomethacin O-demethylation in microsomes of B lymphoblastoid cells expressing human CYPs was investigated at an indomethacin concentration of 5 microM, cDNA-expressed CYP2C9 exhibited 6-fold greater activity than did CYP2C19. At an indomethacin concentration of 50 microM, cDNA-expressed CYP1A2 and CYP2D6 also exhibited slight activities. The KM values were 9.9 +/- 1.2 and 117.1 +/- 13.8 microM and the Vmax values were 0.33 +/- 0.05 and 0.24 +/- 0.04 pmol/min/pmol CYP in microsomes with cDNA-expressed CYP2C9 and CYP2C19, respectively (N = 4). Considering the 16-fold higher intrinsic clearance of CYP2C9, compared with that of CYP2C19, and these expression levels in human livers, the contribution of CYP2C19 to indomethacin O-demethylation was considered to be negligible. Indomethacin appears to be O-demethylated exclusively by CYP2C9 in humans.