BACKGROUND: Aim of this work was to improve standardization of the 13C-urea breath test (13C-UBT) by evaluating the effect of drug administration, feeding and colonic ureolysis on the UBT results. METHODS: Three different studies were performed. First, a 13C-UBT was performed in 41 Helicobacter pylori infected subjects before and after 1 day of therapy with sucralfate (5 g), colloidal bismuth (600 mg), amoxycillin (2.5 g) or omeprazole (40 mg). Second, a 13C-UBT was performed in 10 H. pylori-positive patients after an overnight fast and repeated 2 h after lunch. Finally, a 13C-UBT was carried out in seven healthy volunteers, with breath sampling prolonged to 6 h to investigate colonic bacterial ureolysis. RESULTS: Even a short course of drugs specific for H. pylori may result in a falsely negative 13C-UBT. Feeding does not interfere with the 13C-UBT in infected subjects. No significant 13C-urea breakdown by colonic bacteria is observed during the 13C-UBT when it is prolonged to 6 h. CONCLUSION: The 13C-urea breath test is a sensitive clinical tool for the non-invasive diagnosis of H. pylori infection. It is unaffected by feeding or colonic ureolysis. However, false negative results are likely even after 1 day of therapy with bactericidal, 'cytoprotective' or antisecretory drugs.