In this paper we describe the kinetics of formation of 1-methylxanthine (1-MX), 3-methylxanthine (3-MX) and 1,3-dimethyluric acid (1,3-DMU) from theophylline in human liver microsomal incubations and use the selective inhibitor approach to define the role of the individual cytochrome P450s (CYP) in each pathway. A biphasic model fitted the data best for the formation of each metabolite. The high-affinity site Km and Vmax values were: 1-MX, Km = 0.29 +/- 0.21 mM, Vmax = 5.92 +/- 3.74 pmol.mg(-1).min(-1) (mean +/- S.D.; n = 4); 3-MX, Km = 0.28 +/- 0.08 mM, Vmax = 3.32 +/- 2.19 pmol.mg(-1).min(-1); 1,3-DMU,Km = 0.31 +/- 0.14 mM, Vmax = 43.3 +/- 9.3 pmol.mg(-1).min(-1). The relative contribution of the high- and the low-affinity enzymes in 1,3-DMU formation was calculated based on the enzyme kinetic parameters. To characterize the high-affinity site, a range of CYP isozyme substrates and inhibitors were incubated with 100 microM theophylline. The CYP1A2 inhibitors furafylline, ellipticine and alpha-naphthoflavone were potent inhibitors of both 1-MX and 3-MX formation with more that 80% of N-demethylase activities inhibited below a concentration of 5 microM. These compounds also markedly inhibited 1,3-DMU formation. Enzyme kinetic and selective inhibition data indicated that about 80% of 1,3-DMU formation was catalyzed by the high-affinity isoform (CYP1A2) at a theophylline concentration of 100 microM. To investigate the role of other isoforms in 8-hydroxylation, experiments were performed involving incubation with a combination of inhibitors. It is evident that in addition to CYP1A2, CYP2E1 has a minor role om 8-hydroxylation. This based on the fact that 80% inhibition was seen on preincubation with furafylline and about 90% inhibition on preincubation with furafylline plus diethyldithiocarbamate. Low concentrations of ketoconazole (selective for CYP3A4) only produced marginal inhibition of 1,3-DMU and, therefore, CYP3A4 is only of minor significance in this reaction. Human B-lymphoblastoid cell lines expressing CYP1A2 catalyzed theophylline metabolism with formation of 1-MX, 3-MX and 1,3-MDU. CYP2E1 cells also catalyzed formation of 1,3-DMU. The CYP3A4 cell line did not catalyze theophylline metabolism.