The cytochrome P450s responsible for the regio- and stereoselectivity in the 2- and 3-hydroxylation of the chiral non-steroidal antiinflammatory drug ibuprofen were characterized in human liver microsomes. The rates of formation of both the 2- and 3-hydroxy metabolites exhibited monophasic (N = 2; N is the number of microsomal preparations) and biphasic (N = 2) substrate concentration dependence for both enantiomers of ibuprofen. The high affinity enzyme class parameters for S-ibuprofen (N = 4) were: 2-hydroxylation, Vmax = 566 +/- 213 pmol/min/mg, Km = 38 +/- 13 microM; 3-hydroxylation, Vmax = 892 +/- 630 pmol/min/mg, Km = 21 +/- 6 microM. For R-ibuprofen, the corresponding parameters were: 2-hydroxylation, Vmax = 510 +/- 117 pmol/min/mg, Km = 47 +/- 20 microM; 3-hydroxylation, Vmax = 593 +/- 113 pmol/min/mg, Km = 29 +/- 8 microM. cDNA-expressed CYP2C9 (Arg 144 and Cys 144) favored S-2- and S-3-hydroxyibuprofen formation, but CYP2C8 favored R-2-hydroxyibuprofen formation. Sulfaphenazole, retinol, and arachidonic acid competitively inhibited the rate of formation of all hydroxyibuprofens; Ki values (N = 3) for sulfaphenazole on the 2- and 3-hydroxylations of S-ibuprofen were 0.12 +/- 0.05 and 0.07 +/- 0.04 and of R-ibuprofen were 0.11 +/- 0.07 and 0.06 +/- 0.03 microM, respectively. Sulfaphenazole also competitively inhibited ibuprofen hydroxylation by cDNA-expressed CYP2C9 (Arg 144 and Cys 144) with Ki values in the range of 0.05 to 0.18 microM and CYP2C8 in the range of 0.36 to 0.55 microM. In a bank of 14 human liver microsome samples, significant correlations (r = 0.72 to 0.90; P < 0.01) were observed between the rates of formation of all four hydroxyibuprofens, and for each hydroxyibuprofen and prototypical CYP2C8/9 biotransformations. The regio- and stereoselectivities observed in vitro were consistent with those noted in vivo. The relative levels of both CYP2C8 and CYP2C9 and the expression of the corresponding variants may influence the disposition of ibuprofen in vivo.