Objective: Otitis media is the most commonly diagnosed disease in ambulatory care and Streptococcus pneumoniae continues to be the most common bacterial agent. Bacterial resistance to antibiotics underscores the need for better vaccines. Current pneumococcal conjugate vaccines are modestly protective against otitis media; however, limited serotype coverage and serotype replacement have led to the investigation of pneumococcal proteins as potential vaccine candidates. Two proteins, pneumococcal surface proteins A (PspA) and C (PspC) are important virulence factors, expressed by virtually all strains. Although a number of pneumococcal proteins have been investigated in other infection sites, these proteins can have diverse organ-specific effects. In this study, we investigated the viability and virulence of single (PspA- and PspC-) and double (PspA-/PspC-) mutants of pneumococcal PspA and PspC proteins in the chinchilla middle ear. Methods: Bullae of 24 chinchillas were inoculated with 0.5ml of 106 colony forming units (CFUs)/ml bacteria: 6 with wild-type D39 strain; 6 with PspA-; 6 with PspC-; and 6 with PspA-/PspC- isogenic mutant strains. Bacterial CFU levels in middle ear effusions and light microscopic analysis of the number of inflammatory cells in the round window membrane (RWM) were compared 48h after inoculation. Results: At 48h, CFUs in middle ears were increased for wild-type and PspC- strains compared to inoculum levels; however, they were significantly less for the group inoculated with the PspC- strain compared to wild-type strain. No bacteria were detected in the PspA- and PspA-/PspC- groups. The number of inflammatory cells in the RWM was significantly higher in wild-type compared to the PspA-, PspC-, and PspA-/PspC- groups. No significant difference in number of inflammatory cells was observed between any pairs of groups inoculated with mutant strains. Conclusion: Viability and virulence of the PspC- strain were similar to the wild-type strain. The single PspA- and double PspA-/PspC- mutants were highly attenuated in the ear. Bacterial clearance of the PspA-/PspC- double mutant was indistinguishable from that of the PspA mutant. These studies provide no reason to exclude PspC from a multi-component protein vaccine containing PspA. © 2014 Elsevier Ireland Ltd.