A high performance liquid chromatographic (HPLC) technique for the determination of three metabolites of theophylline, 3-methylxanthine (3-MX), 1-methylxanthine (1-MX) and 1,3-dimethyluric acid (1,3-DMU) in human liver microsomes is described. The analytes were extracted from human liver microsomes with methylene chloride/isopropanol and stepwise gradient elution was employed for the resolution of peaks. The limits of quantitation were 15 ng/mL for 3-MX, 20 ng/mL for 1-MX and 20 ng/mL for 1,3-DMU. The calibration range was linear for the three metabolites and the calibration ranges were 15-250 ng/mL for 3-MX, 20-250 ng/mL for 1-MX and 250-4000 ng/mL for 1,3-DMU. The absolute recovery ranged from 63-84% for 3-MX, 65-79% for 1-MX and 77-89% for 1,3-DMU over the calibration curve range. Accuracy for all three metabolites was within +/- 10% and adequate selectivity was demonstrated by the lack of interfering peaks in blank chromatograms. The within-run and interday precision were within 10% RSD for all three metabolites tested at two concentrations. The advantage of this method over previous methods is that the use of quaternary ammonium ion pair reagents in the mobile phase has been obviated. Also, unlike a previous radiometric HPLC method, the need for radiolabelled theophylline has also been eliminated. The method was used to characterize theophylline metabolism in human liver microsomes for immunoinhibition studies and to investigate the interaction of theophylline with selected quinolone antibiotics.