Several diagnostic assays are available for evaluating Helicobacter pylori infection: histological examination, culture of gastric biopsies, urea breath test and serology. Recently a new enzyme immunoassay has been introduced for the detection of H. pylori antigens in stool samples (HpSA). The aim of our study was to evaluate and compare diagnostic efficacy of HpSA with histological examination, culture, urea breath test and serology in a group of 95 patients. Patients were classified H. pylori positive (43) or negative (52) on the basis of histology, culture and urea breath test. HpSA optical densities were significantly higher in infected patients compared to those obtained in H. pylori-negative patients (t = 5.47, p < 0.001). Overall, with a fixed cut-off of 0.1 unit of optical density, the sensitivity was 79% and the specificity 100%. In the H. pylori positive patients, HpSA optical density correlated with bacterial load histologically evaluated in the gastric antrum (r = 0.405, p < 0.05) and was inverse correlated with levels of serum IgG elicited against H. pylori (r = -0.315, p < 0.05). Considering patients with a positive HpSA finding and/or levels of anti-H. pylori antibodies upper than 30 U/mL, sensitivity in detecting infected patients was 98%.(1) immunodetection of H. pylori antigens in stools is a good alternative of breath test; (2) a reduction in H. pylori density grade might be accompanied by low HpSA optical density, leading to a false negative result and (3) combining the HpSA determination with the serum detection of anti-H. pylori antibodies a better clinical sensitivity is obtained.