A 13C-urea breath test for detection of Helicobacter pylori infection was validated in 306 patients. Breath samples (four, two or one) were taken at various time points within 30 minutes after a motility inhibiting liquid test meal with citric acid followed by 75 mg of 13C-urea. The 13CO2/12 CO2-ratio (delta-value) was measured using isotope ratio mass spectrometry and the recovery of tracer in the exhaled breath was calculated (UBT-value). In 172 patients a comparison of established reference methods (culture, CLO test, and Fuchsin staining) with the 4 point breath analysis for detection of Helicobacter pylori showed high values of quality parameters (greater than 80% for sensitivity, specificity, positive and negative predictive value), independent of expression of the results as delta-value or as UBT-value. In 134 patients a reduction of samples to one single breath sample taken 30 minutes after ingestion of the tracer showed no significant differences in the quality parameters when compared with the standard 4 point breath analysis. This indicates that the analysis of a single breath sample is suitable for detection of Helicobacter pylori status in man.