Myeloid cells, including neutrophils and macrophages, play important roles in innate immune defense against acute bacterial infections. Myeloid Src family kinases (SFKs) p59/61(hck) (Hck), p58(c-fgr) (Fgr), and p53/56(lyn) (Lyn) are known to control integrin beta(2) signal transduction and FcgammaR-mediated phagocytosis in leukocytes. In this study, we show that leukocyte recruitment into the cerebrospinal fluid space and bacterial clearance is hampered in mice deficient in all three myeloid SFKs (hck(-/-)fgr(-/-)lyn(-/-)) during pneumococcal meningitis. As a result, the hck(-/-)fgr(-/-)lyn(-/-) mice developed increased intracranial pressure and a worse clinical outcome (increased neurologic deficits and mortality) compared with wild-type mice. Impaired bacterial killing was associated with a lack of phagocytosis and superoxide production in triple knockout neutrophils. Moreover, in hck(-/-)fgr(-/-)lyn(-/-) neutrophils, phosphorylation of p40(phox) was absent in response to pneumococcal stimulation, indicating a defect in NAPDH oxidase activation. Mice lacking the complement receptor 3 (CR3; CD11b/CD18), which belongs to the beta(2)-integrin family, also displayed impaired host defense against pneumococci, along with defective neutrophil superoxide production, but cerebrospinal fluid pleocytosis was normal. Cerebral expression of cytokines and chemokines was not decreased in both mouse strains, indicating that CR3 and myeloid SFKs are dispensable for the production of inflammatory mediators. Thus, our study demonstrates the pivotal role of myeloid SFKs and CR3 in mounting an effective defense against CNS infection with Streptococcus pneumonia by regulating phagocytosis and NADPH oxidase-dependent superoxide production. These data support the role of SFKs as critical mediators of CR3 signal transduction in host defense.