Results from studies using mice deficient in specific complement factors and clinical data on patients with an inherited deficiency of the classical complement pathway component C2 suggest that the classical pathway is vital for immunity to Streptococcus pneumoniae. However, the consequences of defects in classical pathway activity for opsonization with C3b and the phagocytosis of different S. pneumoniae serotypes in human serum are not known, and there has not been a systematic analysis of the abilities of sera from subjects with a C2 deficiency to opsonize S. pneumoniae. Hence, to investigate the role of the classical pathway in immunity to S. pneumoniae in more detail, flow cytometry assays of opsonization with C3b and the phagocytosis of three capsular serotypes of S. pneumoniae were performed using human sera depleted of the complement factor C1q or B or sera obtained from C2-deficient subjects. The results demonstrate that, in human serum, the classical pathway is vital for C3b-iC3b deposition onto cells of all three serotypes of S. pneumoniae and seems to be more important than the alternative pathway for phagocytosis. Compared to the results for sera from normal subjects, C3b-iC3b deposition and total anti-S. pneumoniae antibody activity levels in sera obtained from C2(-/-) subjects were reduced and the efficiency of phagocytosis of all three S. pneumoniae strains was impaired. Anticapsular antibody levels did not correlate with phagocytosis or C3b-iC3b deposition. These data confirm that the classical pathway is vital for complement-mediated phagocytosis of S. pneumoniae and demonstrate why subjects with a C2 deficiency have a marked increase in susceptibility to S. pneumoniae infections.