The development of metronidazole resistance in Helicobacter pylori has become an important factor in the clinical use of this drug in the treatment of gastritis and peptic ulcers. The mechanism for the development of resistance is not known but may involve factors which influence metronidazole uptake and metabolism. In this study, a simple and reliable HPLC assay for metronidazole was developed and the uptake of the drug from broth cultures of both resistant and sensitive strains of H. pylori was investigated. Resistance to metronidazole was determined by the E test as well as plate and broth assays. In the presence of the antibiotic (approximately 10 mg/L), metronidazole-sensitive strains were killed within 24 h and there was a concomitant fall in the concentration of metronidazole in the broth. In contrast, the killing of resistant strains of H. pylori was associated with < 10% loss of antibiotic from broth. These differences in metronidazole uptake are ascribed to alterations in the mechanisms of action of the drug in sensitive and resistant strains.