A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of theophylline(TP) and its metabolites, 1-methyluric acid (1-MU) and 1,3-dimethyluric acid (1,3-DMU), in rat plasma and urine. An $100;{mu}l$ aliquot of a plasma or urine sample was mixed with $250;{mu}l$ of acetonitrite and vortexed. After centrifugation, $200;{mu}l$ (plasma) or $20;{mu}l$ (urine) aliquot of the supernatant was dried by $N_2$ stream and redissolved in $100;{mu}l$ (plasma) or $200;{mu}l$ (urine) of the mobile phase. A $20;{mu}l$ of the mobile phase solution was injected onto a $C_{18}$ reversed-phase column. The column was maintained at $45^{circ}C$ by the aid of electric heating jacket. The mobile phase was a 3%(v/v) methanol solution in deionized water which contains sodium acetate (100 mM) and tetrabutyl ammonium hydroxide (4 mM). pH of the mobile phase was adjusted 4.5 by the addition of acetic acid. Detection limits for TP, 1-MU, and 1,3-DMU in plasma were 0.2, 0.1 and $0.1;{mu}/ml$, respectively and the corresponding values in urine were all $5;{mu}g/ml$. Inter- and intra-day variability of the assay for all compounds in the plasma samples was less than 5.5 and 3.8%, respectively. The retention times for 1-MU, 1,3-DMU, and TP were approximately 7, 8.5 and 18 min, respectively. Sample preparation procedure used in this method was simple, rapid and reproducible. Renal clearance of TP and its metabolites in rats showed plasma concentration dependency indicating renal tubular secretion and reabsorption of them.