Host immune factors involved in bacterial ocular virulence were examined through the use of pneumolysin, a cytoplasmic protein and pneumococcal strain D39 utilizing human corneal epithelial cells (HCEC). Recombinant pneumolysin activity was determined by a hemolysis assay using the following concentrations: 50-1000 ng/mL. HCEC were grown to confluency and exposed to 10³ to 10⁷ colony forming units (CFUs) of mid-log phase Streptococcus pneumoniae D39 for 2, 4, 6, and 24 h. HCEC were exposed to pneumolysin at various concentrations: 200-1000 ng/mL. ELISA and human cytokine array was performed on the supernatant. Fluorescence studies using cytotoxicity assay were performed on the HCEC cells following infection to determine live versus dead cells. The hemolysis assay showed that a minimum of 100 ng/mL PLY will lyse the red blood cells. Cytokine array on the HCEC exposed supernatants showed the presence of IL-6 and IL-8. Four hour exposure of HCEC cells to a range of 10³ to 10⁷ CFU of D39 resulted in monolayer disruption and decreased viability as compared to cells exposed to the media alone. HCEC cells exposed to any of the concentrations of bacteria exhibited signs of cellular damage and death. Following 24 h exposure, there was an increased expression of IL-8 (p < 0.001) compared to media alone. IL-6, IL-8 and IFN-gamma levels were elicited at a minimal of 200ng PLY. In addition to changes in viability and morphology of cultured cells, we detected specific cytokines in the culture supernatants of HCEC following exposure to S. pneumoniae D39.