Streptococcus pneumoniae infections of the lung are associated with significant damage to the alveolar epithelium. Host phagocytes and pneumolysin, a cytolytic toxin of S. pneumoniae, are believed to contribute to this cellular damage, yet experiments in which these elements are absent demonstrate the presence of an additional soluble S. pneumoniae factor that is toxic to alveolar epithelium. We examined the effects of S. pneumoniae-associated alveolar epithelial cell injury by factors other than S. pneumoniae-derived pneumolysin or phagocyte products by exposing cultured rat type II alveolar epithelial cells (RAEC) to S. pneumoniae mutants that lacked pneumolysin activity. We found that mutant pneumolysin-deficient strains of S. pneumoniae produced injury to RAEC similar to that produced by the parent strains. A toxin of type 14 S. pneumoniae was distinguished from pneumolysin by physiochemical (i.e., molecular mass and heat stability) and functional (i.e., hemolytic activity and cytotoxic activity) properties and was identified as hydrogen peroxide. All S. pneumoniae strains tested produced hydrogen peroxide, and in many strains hydrogen peroxide production was comparable to that of activated neutrophils. We conclude that S. pneumoniae produces hydrogen peroxide in concentrations that are cytotoxic to RAEC in vitro and that alveolar epithelial damage due to hydrogen peroxide may be involved in the pathogenesis of host cellular injury in pneumococcal pneumonia.