Type I and type II interferons (IFNs) act in synergy to inhibit the replication of a variety of viruses, including herpes simplex virus (HSV). To understand the mechanism of this effect, we have analyzed the transcriptional profiles of primary human fibroblast cells that were first treated with IFN-beta1, IFN-gamma, or a combination of both and then subsequently infected with HSV-1. We have identified two types of synergistic activities in the gene expression patterns induced by IFN-beta1 and IFN-gamma that may contribute to inhibition of HSV-1 replication. The first is defined as "synergy by independent action," in which IFN-beta1 and IFN-gamma induce distinct gene categories. The second, "synergy by cooperative action," is a term that describes the positive interaction between IFN-beta1 and IFN-gamma as defined by a two-way analysis of variance. This form of synergy leads to a much higher level of expression for a subset of genes than is seen with either interferon alone. The cooperatively induced genes by IFN-beta1 and IFN-gamma include those involved in apoptosis, RNA degradation, and the inflammatory response. Furthermore, the combination of IFN-beta1 and IFN-gamma induces significantly more apoptosis and inhibits HSV-1 gene expression and DNA replication significantly more than treatment with either interferon alone. Taken together, these data suggest that IFN-beta1 and IFN-gamma work both independently and cooperatively to create an antiviral state that synergistically inhibits HSV-1 replication in primary human fibroblasts and that cooperatively induced apoptosis may play a role in the synergistic effect on viral replication.