Nabumetone, a widely used nonsteroidal anti-inflammatory drug, requires biotransformation into 6-methoxy-2-naphthylacetic acid (6-MNA), a close structural analog to naproxen, to achieve its analgesic and anti-inflammatory effects. Despite its wide use, the enzymes involved in metabolism have not been identified. In the present study, several in vitro approaches were used to identify the cytochrome P450 (P450) enzyme(s) responsible for 6-MNA formation. In human liver microsomes (HLMs) 6-MNA formation displayed monophasic Michaelis-Menten kinetics with apparent K(m) and V(max) values (mean +/- S.D.) of 75.1 +/- 15.3 microM and 1304 +/- 226 pmol/min/mg protein, respectively, and formation rate of 6-MNA varied approximately 5.5-fold (179-983 pmol/min/mg protein). 6-MNA activity correlated strongly with both CYP1A2-mediated phenacetin O-deethylation activity and CYP1A2 protein content (r = 0.85 and 0.74, respectively; p < 0.0001 for both). Additional correlations were found with model activities of CYP2C19 and CYP3A4. Of 11 cDNA-expressed recombinant P450s used, recombinant CYP1A2 was the major form catalyzing the 6-MNA formation with an apparent K(m) of 45 microM and V(max) of 8.7 pmol/min/pmol P450. Minor fractions were catalyzed by recombinant P450s CYP1A1, CYP2B6, CYP2C19, CYP2D6, and CYP2E1. Experiments with P450-selective chemical inhibitors and monoclonal anti-P450 antibodies showed that furafylline, a mechanism-based inhibitor CYP1A2, and anti-CYP1A2 antibody markedly inhibited 6-MNA formation, whereas inhibitors for other P450s did not show significant inhibitory effects. Taken together, these studies indicate that the formation of the active metabolite of nabumetone, 6-MNA, is predominantly catalyzed by CYP1A2 in HLMs with only minor contribution of other P450s.