A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small Doudoune Parajumpers Gobi volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm Parajumpers Gobi Eco I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml−1 Manteau Homme Parajumpers for gastric juice aspirate and plasma and 0.010 μg g−1 for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml−1 for plasma and gastric juice aspirate and up to 40 μg g−1 for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.