The role of the cytolytic toxin pneumolysin in the pathogenicity of Streptococcus pneumoniae was investigated. Pneumolysin was purified to homogeneity and used to immunize mice. When these mice were subsequently challenged via the nasal route with virulent S. pneumoniae, they survived significantly longer than control mice. The mean survival times were 5.52 and 2.48 days for immunized and control mice, respectively. This work provides direct evidence for the involvement of pneumolysin in pneumococcal pathogenicity.