To better understand the significance of viral upper respiratory tract infections in the pathogenesis of acute otitis media (OM), 27 adults underwent intranasal inoculation with influenza A virus. Monitoring consisted of antibody titer determination, tympanometry, and otoscopy. Microbiologic analysis consisted of cultures and polymerase chain reaction (PCR)-based detection for influenza A virus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. All subjects became infected with the challenge virus. By day 4, 16 (59%) developed middle ear pressures of -100 mm H2O or below and 4 (25%) of them developed OM. One subject (4%) developed purulent OM requiring myringotomy for pain relief. Middle ear effusion cultures were negative. PCR analysis of that subject's middle ear effusion and nasal washes were positive for influenza A virus and S. pneumoniae. These findings support a causal role for viral upper respiratory tract infections in the pathogenesis of OM, possibly mediated by middle ear underpressures and viral and bacterial middle ear infection.