OBJECTIVES: To confirm the finding of pepsin/pepsinogen in middle ear fluid of children with chronic or recurrent otitis media (OME or ROM), and to ask parents about symptoms associated with gastroesophageal reflux (GER). METHODS: Middle ear fluid was collected from children undergoing tympanostomy tube placement. We tested this fluid for pepsin/pepsinogen using a proteolytic enzyme assay and an ELISA. Parents completed questionnaires about symptoms of GER in their children. RESULTS: We collected 36 samples from 22 children; 16 of 22 children (73%) were positive with the proteolytic assay; 17 (77%) were positive with ELISA. Questionnaires did not show increased GER symptoms. CONCLUSIONS: We replicated the finding of pepsin/pepsinogen in middle ear fluid of children with OME or ROM, but did not find any increase in GER symptoms. Further research is needed to establish a causative link between GER and OM.