Ebrotidine is a new H2-receptor antagonist with powerful antisecretory activity, demonstrated gastroprotection and the ability to inhibit protease and lipase activities of Helicobacter pylori. As a tool in the clinical pharmacokinetic study of ebrotidine, an analytical method for the simultaneous determination of ebrotidine an its metabolites in human urine was developed. An ion-pair reversed-phase HPLC separation using 1-hexanesulfonic acid and acetonitrile as mobile phase with gradient elution was optimized. In addition, several procedures of preconcentration and clean-up were tested, including solid-phase and liquid-liquid extraction, the mixture dichloromethane-2-propanol (9:1, v/v) at pH 11 being the most efficient. The quality parameters of the whole analytical method were established, the calibration curves were linear over the range studied (1-200 micrograms/ml) and the reproducibility of the method was high (inter-day R.S.D. values lower than 4.4%). The limits of detection were between 26 and 110 ng/ml of urine for ebrotidine and its metabolites. The method was applied to the analysis of urine collected from two volunteers during 96 h following oral administration of ebrotidine at a dose of 400 mg.