In vivo evidence suggests that T-cell-derived gamma interferon (IFN-gamma) can directly inhibit the replication of herpes simplex virus type 1 (HSV-1). However, IFN-gamma is a weak inhibitor of HSV-1 replication in vitro. We have found that IFN-gamma synergizes with the innate IFNs (IFN-alpha and -beta) to potently inhibit HSV-1 replication in vitro and in vivo. Treatment of Vero cells with either IFN-beta or IFN-gamma inhibits HSV-1 replication by <20-fold, whereas treatment with both IFN-beta and IFN-gamma inhibits HSV-1 replication by approximately 1,000-fold. Treatment with IFN-beta and IFN-gamma does not prevent HSV-1 entry into Vero cells, and the inhibitory effect can be overcome by increasing the multiplicity of HSV-1 infection. The capacity of IFN-beta and IFN-gamma to synergistically inhibit HSV-1 replication is not virus strain specific and has been observed in three different cell types. For two of the three virus strains tested, IFN-beta and IFN-gamma inhibit HSV-1 replication with a potency that approaches that achieved by a high dose of acyclovir. Pretreatment of mouse eyes with IFN-beta and IFN-gamma reduces HSV-1 replication to nearly undetectable levels, prevents the development of disease, and reduces the latent HSV-1 genome load per trigeminal ganglion by approximately 200-fold. Thus, simultaneous activation of IFN-alpha/beta receptors and IFN-gamma receptors appears to render cells highly resistant to the replication of HSV-1. Because IFN-alpha or IFN-beta is produced by most cells as an innate response to virus infection, the results imply that IFN-gamma secreted by T cells may provide a critical second signal that potently inhibits HSV-1 replication in vivo.