INTRODUCTION: Helicobacter pylori infection affects more than half the world's population. It is a major cause of chronic gastritis and there is a strong association with peptic ulceration and gastric adenocarcinoma. Rifaximin is a new nonabsorbable broad-spectrum antimicrobial agent that reaches high concentrations in the gastrointestinal tract. AIM: To evaluate the in vitro activity of rifaximin against H. pylori isolates. METHODS: Thirty-one H. pylori strains were analyzed by the agar dilution method. Clarithromycin was used as the control antibiotic. Staphylococcus aureus and Streptococcus pneumoniae were used as quality control strains. Plates were read at days 4 and 7 of incubation. The MIC50 and MIC90 of each antibiotic were calculated. Strains with a clarithromycin MIC of > 1 μg/ml were considered resistant. RESULTS: The MIC50 of clarithromycin at days 4 and 7 was 0.125 μg/ml and the MIC90 at days 4 and 7 ranged from 8 to 16 μg/ml, respectively. The MIC50 of rifaximin at days 4 and 7 ranged from 1 to 2 μg/ml, respectively, and the MIC90 was 4 μg/ml at both days 4 and 7. Twenty percent of H. pylori strains were resistant to clarithromycin. All clarithromycin-resistant strains were inhibited at a maximal rifaximin concentration of 4 μg/ml. CONCLUSION: These results indicate that this new antibiotic may be useful for eradication of A pylori infection. Because rifaximin is active against H. pylori strains resistant to clarithromycin, it could be useful in combination with this drug or in the treatment of therapeutic failure.