Pneumolysin, the major Streptococcus pneumoniae cytotoxin, contributes to the early pathogenesis of invasive pneumococcal pneumonia by facilitating intrapulmonary bacterial growth and invasion into the blood. Pneumolysin is a multifunctional toxin, with distinct cytolytic ("hemolytic") and complement-activation ("complement") activities that have been mapped to several regions of the molecule. To characterize the specific contributions of pneumolysin's hemolytic and complement properties to the pathogenesis of pneumococcal pneumonia, we compared the in vivo effects of type 2 S. pneumoniae mutant strains, which produce pneumolysins deficient in these activities. The absence of either pneumolysin's hemolytic or complement activities rendered mutant strains less virulent than the wild-type strain during pulmonary infection. Pneumolysin's hemolytic activity correlated with acute lung injury and bacterial growth at 3 and 6 h after endotracheal instillation. In contrast, pneumolysin's complement activity correlated with bacterial growth and bacteremia at 24 h after pulmonary infection. Pneumolysin's complement activity was not associated with the degree of alveolar-capillary injury or recruitment of leukocytes during initial pulmonary infection. However, pneumolysin's complement activity inhibited killing of mutant bacteria in an in vitro complement-dependent neutrophil killing assay. Thus, both pneumolysin's hemolytic and complement activities made specific contributions to the early pathogenesis of pneumococcal pneumonia at different stages of infection and by different mechanisms.