ABSTRACT The initiation of the immune response at the cellular level relies on specific recognition molecules to rapidly signal viral infection via interferon (IFN) regulatory factor 3 (IRF-3)-dependent pathways. The absence of IRF-3 would be expected to render such pathways inoperative and thereby significantly affect viral infection. Unexpectedly, a previous study found no significant change in herpes simplex virus (HSV) pathogenesis in IRF-3 −/− mice following intravenous HSV type 1 (HSV-1) challenge (K. Honda, H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, and T. Taniguchi, Nature 434:772-777, 2005). In contrast, the present study demonstrated that IRF-3 −/− mice are significantly more susceptible to HSV infection via the corneal and intracranial routes. Following corneal infection with 2 × 10 6 PFU of HSV-1 strain McKrae, 50% of wild-type mice survived, compared to 10% of IRF-3-deficient mice. Significantly increased viral replication and inflammatory cytokine production were observed in brain tissues of IRF-3 −/− mice compared to control mice, with a concomitant deficit in production of both IFN-β and IFN-α. These data demonstrate a critical role for IRF-3 in control of central nervous system infection following HSV-1 challenge. Furthermore, this work underscores the necessity to evaluate multiple routes of infection and animal models in order to fully determine the role of host resistance factors in pathogenesis.