ABSTRACT To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 ( Bf/C2 −/ − ), C1qa ( C1qa −/ − ), and factor B ( Bf − / − ). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2 −/ − , Bf − / − , and C1qa −/ − mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection ( P < 0.05) and remained persistently higher in samples from Bf/C2 −/ − mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2 −/ − , Bf − / − , and C1qa −/ − mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2 −/ − mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa − / − and Bf − / − mice, and least for Bf/C2 − / − mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae . The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.