Rapid diagnostic tools for Helicobacter pylori are important in endoscopy. To assess the accuracy of a new 5-min rapid urease test (UFT300, ABS Cernusco, sul Naviglio, Italy) compared with the 1-h Pyloritek (Serim Laboratories, Elkhart, IN) and the 24-h CLO test (Kimberly-Clark Ballard Medical Products, Roswell, GA), consecutive dyspeptic patients referred to our unit for endoscopy were prospectively studied. All patients underwent a 13C-urea-breath test, histology and the UFT300 (ABS; Cernusco, sul Naviglio, Italy). Two additional rapid urease tests were performed. Patients were deemed infected when both 13C-UBT and histology were positive. Rapid urease tests were read at 1, 5, and 60 min, respectively. Of the 375 enrolled patients, 45.3% were infected with H. pylori. The sensitivity of the new 5-min rapid urease test 300 was 90.3, 94.5, and 96.2% at 1, 5, and 60 min, respectively (specificity 100%). The Pyloritek and the new 5-min rapid urease test were comparable, but the CLO test was not reliable at 5 and 60 min. In conclusion, the new 5-min rapid urease test is comparable to the Pyloritek test, but the CLO test is significantly less sensitive at early time points. Reading the test results at 1 min may increase false-negative results with decreasing sensitivity.