The detection of immunostimulatory DNA is well documented to occur at several cellular sites, but there is limited evidence of nuclear innate DNA sensing. Prior to this study, the detection of herpesviral DNA was thought to be restricted to the cytosol so as to limit the sensing of host DNA in the nucleus. However, given the nuclear lifecycle of these viruses, we hypothesized that viral DNA could be sensed in the nucleus of infected cells. To test this hypothesis we examined the activation of interferon regulatory factor 3 (IRF-3) in response to herpes simplex virus 1 (HSV-1) infection of primary human foreskin fibroblasts (HFF). Using a mutant defective for expression of all viral genes, we observed that the release of viral DNA into the nucleus is necessary to activate IRF-3 signaling. Furthermore, we determined this response to be dependent on nuclear-localized interferon inducible protein 16 (IFI16) and the cytoplasmic stimulator of interferon genes (STING) adaptor protein.