Toll-like receptor 3 (TLR-3) and TLR-9 gene expression and interleukin 6 (IL-6) secretion were studied in corneal cells with components of herpes simplex virus (HSV). Human corneal epithelial cells (HCEs) and primary human corneal fibroblasts (HCRFs) were infected with live HSV or UV-inactivated HSV (UV-HSV), transfected with HSV DNA or treated with HSV-anti-HSV IgG immune complexes. Gene expression of TLR-3 and -9 was analysed by real-time PCR. Supernatants were assayed for IL-6 by ELISA. Incubation of HCEs and HCRFs with live HSV-1, UV-HSV and HSV DNA resulted in augmented TLR-3 and -9 gene expression and IL-6 release. Moreover, infected or transfected HCRFs released greater amounts of IL-6 than did HCEs. As virus is frequently in the form of neutralized virus immune complexes, the ability of these immune complexes to interact with TLRs and trigger IL-6 production was evaluated. Here, it is shown that HSV-anti-HSV IgG complexes were as potent as HSV DNA in their ability to induce IL-6. Treatment of HCRFs transfected with HSV DNA with the TLR-9-inhibitory oligomer iODN, anti-TLR-3 antibody or phosphatidylinositol 3-kinase inhibitor indicated that IL-6 release from HCRFs was mediated by TLR-3 and -9 gene expression. These results demonstrated that neutralized HSV immune complexes were as potent as HSV DNA in enhancing IL-6 release from corneal fibroblasts. These phenomena were mediated via augmented TLR-3 and -9 gene expression.