Objective: To determine whether mutants of Streptococcus pneumoniae that are deficient in pneumococcal surface protein A (PspA), pneumococcal surface antigen A (PsaA), or pneumolysin (Ply) are less virulent and less likely to penetrate the round window membrane (RWM). Design: Histopathologic comparison of wild-type S pneumoniae and its mutants deficient in PspA, PsaA, and Ply. Setting: Otopathology Laboratory, Department of Otolaryngology, University of Minnesota Medical School, Minneapolis. Participants: Forty young chinchillas (weight, 250-350 g) with normal external auditory canals and tympanic membranes. Intervention: Animals were divided into 3 groups and bullae inoculated with wild-type S pneumoniae serotype 2, strain D39, or its mutants deficient in PspA, PsaA, or Ply. Two days after inoculation, bullae were processed for light microscopy and transmission electron microscopy. Main Outcome Measures: Comparison of inflammatory cell infiltration and penetration of bacteria into the round window membrane and adjacent scala tympani. Results: Histopathologic findings using wild-type S pneumoniae and Ply- mutant were similar and included otitis media and the presence of inflammatory cells and damage to and passage of bacteria through the RWM. Although otitis media was seen with the PspA- and PsaA- mutants, we observed no passage of bacteria through the RWM. Conclusions: Both PspA and PsaA affect the ability of S pneumoniae to penetrate the RWM. Understanding the role of S pneumoniae virulence proteins in the pathogenesis of the middle ear, RWM, and inner ear will provide new strategies for the prevention and treatment of otitis media and its complications. ©2008 American Medical Association. All rights reserved.