In earlier experiments on the role of the cytolytic toxin pneumolysin in ocular infections with pneumococcus, we found that a strain carrying a deletion in the gene encoding pneumolysin was considerably less virulent than wild type when tested in an intracorneal model of keratitis in the rabbit. To confirm this result, we have constructed a strain in which pneumolysin activity was restored by transformation of the deleted strain with a plasmid bearing the complete pneumolysin gene. Hemolytic titers of pneumolysin indicated that only one copy of the plasmid per bacterium expresses the pneumolysin gene in this strain. The virulence of this strain was compared with that of wild type and deleted strains transformed with the vector lacking the pneumolysin gene. Slit lamp examination (SLE) scores for eyes infected with the restored strain were similar to those for eyes infected with wild type and significantly greater than those for the pneumolysin-deleted strain. Molecular analysis of bacteria recovered from infected corneas showed that the vector plasmid was retained; however, in most isolates of the restored strain, the plasmid underwent an excision and lost the pneumolysin gene. The cloned gene apparently persisted long enough to induce the pathologic changes, and the results confirm the importance of pneumolysin as a virulence factor in ocular infections.