Culture and susceptibility testing of Helicobacter pylori strains was performed in a large multinational, multicenter randomized clinical trial. Culture was carried out on gastric biopsy samples obtained from 516 patients at entry and had a sensitivity of 99% when the [(13)C]urea breath test was used as a reference. Susceptibility testing was performed for clarithromycin and metronidazole on 485 strains by an agar dilution method and the epsilometer test (Etest) and for amoxicillin by an agar dilution method only. Resistance to clarithromycin (>1 microgram/ml) was found in 3% of the H. pylori strains, with a perfect correlation between Etest and agar dilution methods. Resistance to metronidazole (>8 microliter/ml) was found in 27% of the strains by agar dilution, but there were important discrepancies between it and the Etest method. No resistance to amoxicillin was found. The logarithms of the MICs of the three antibiotics against susceptible strains had a distribution close to normal. The impact of resistance was tested in the four arms of the trial. There were not enough clarithromycin-resistant strains to evaluate the impact of resistance on the cure rate of clarithromycin-based regimens. For metronidazole-resistant strains, the impact noted in the clarithromycin-metronidazole arm was partially overcome when omeprazole was added (76% eradication for resistant strains versus 95% for susceptible strains). Secondary resistance to clarithromycin occurred in strains from 12 of 105 patients (11.4%) after the failure of a clarithromycin-based regimen to effect eradication. The detection of point mutations in clarithromycin-resistant strains was performed by a combination of PCR and restriction fragment length polymorphism. Mutations (A2142G and 2143G) were found in all strains tested except one. This study stresses the importance of performing susceptibility tests in clinical trials in order to explain the results of different treatments.