A simple and reproducible method for the determination of cimetidine in serum is described. Separation and quantitation were performed by high-performance liquid chromatography using a Radial-Pak CN column with a mobile phase of 33 % acetonitrile solution containing 5 mM triethylamine (adjusted to pH 3.0 with phosphoric acid) at a flow rate of 2 ml/min and at a detection of 220 nm. Cimetidine and ranitidine as an internal standard were extracted from serum with ethyl acetate and then back-extracted into dilute acid. An aliquot of the dilute acid was analyzed in the chromatographic system. The limit of detection was as low as 0.02 μg/ml using 100 μl of serum at a signal-to-noise ration of 2. Recoveries of cimetidine and ranitidine from serum were both greater than 95%. Within-run and day-to-day reproducibility for 10.0 μg/ml samples were 2.6 % and 2.9 %, respectively. The method is applicable to pharmacokinetic studies.