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In the eradication therapy of Helicobacter pylori changes of antibiotics as these concentrations or amount in the stomach after oral administration were not clear. A simple and accurate method for determining the concentration of metronidazole (MTZ) in homogenate of rat stomach was developed in order to obtain basic data to design a pharmaceutical preparation having targeting ability to the surface of gastric-mucosa. This method included a deproteinization process by methanol, separation with reversed-phase high-performance liquid chromatography, and detection with an ultraviolet wavelength of 370 nm. Regression analysis showed that the method was linear over a standard curve nmge from 5 ug/mL to 2000 g/mL. The inter-day precision and accuracy values between thc ranges were 5.0% or better and -7.5 to 5.2%, respectively. The newly developed method was applied to an analysis of gastric samples after oral administration of MTZ at a dose of 5 mg/kg. It was found that the residual MTZ in the stomach was determined within 5 h after dosing. This method is useful for monitoring MTZ in stomach after its oral administration to rats.