OBJECTIVE Two Streptococcus pneumoniae proteins, pneumococcal surface protein A (PspA) and pneumolysin (Ply), have functional and histopathologic effects on the inner ear. BACKGROUND Temporary or permanent sensorineural hearing loss is known to be a sequela of pneumococcal otitis media. Several pneumococcal proteins such as PspA and Ply have been shown to contribute to the pathogenesis of the middle ear; however, effects of these proteins on the inner ear and hearing loss are unknown. METHODS Middle ears of chinchillas were inoculated with either wild-type S. pneumoniae or its mutants, deficient in PspA or Ply proteins. After 28 days, auditory brainstem response of animals was tested, and their bullae were processed for histopathologic analysis by light microscopy. RESULTS Twenty-eight days after instillation of 20 colony-forming units of wild-type pneumococci, auditory brainstem response test showed threshold changes of 10 to 15 dB for 4 to 32 kHz and more than 20 dB for 1 to 2 kHz. No significant hearing loss was observed after instillation of the same or even higher doses of isogenic S. pneumoniae mutants of PspA or Ply proteins, or saline injection, after the same period. Histologic analysis showed no fluid, inflammatory cells, or bacteria in the middle ear, indicating that hearing loss was sensorineural. Inner ear morphology showed pathologic changes in the stria vascularis, suggesting it as the target of otitis media-induced damage, which may lead to sensorineural hearing loss. CONCLUSIONS The virulence PspA and Ply proteins of S. pneumoniae affect the inner ear and auditory function.