Objective: The aim of this study was to investigate the presence of Helicobacter pylori by CLO test in the middle ear of the patients with chronic otitis media. We also investigated the relationship between H. pylori in the stomach and in the middle ear by C-14 urea breath test (C-14 UBT) for the possible source of this bacterium. Study Design: A prospective controlled clinical study. Methods: Tissue samples were obtained from the middle ear mucosa of 41 patients undergoing ear surgery for chronic suppurative otitis media and placed in the CLO test kit. C-14 UBT was performed in 24 patients and 20 normal subjects. Results: The CLO test results were positive in 22 patients (53.6%). Results of C-14 UBT that was performed in 24 patients were positive in 19 patients (79.1%) and were positive in 6 control subjects (30%). Conclusion: Fifty-three percent of the middle ear mucosae obtained from 41 patients with chronic otitis media were shown to be positive for H. pylori, and although our results suggest that the source of this bacterium is the stomach, further clinical studies are needed.