Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.