Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-/]) and type II interferon (IFN-) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-upon macrophage activation with IFN-is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-. Murine cytomegalovirus (MCMV) infection in mice is a well-established model system for the study of acute, persistent , and latent infections of betaherpesviruses and their control by the host immune system (32, 35, 58, 60). Both human CMV (HCMV) and MCMV infect a broad range of tissues in their respective hosts, including fibroblasts, endothelial and epithelial cells, and, significantly, immune cells of the myeloid lineage (13, 57, 69, 70). Differentiated macrophages of this lineage residing in infected tissues play a key role in eliciting the host immune response but are also permissive for CMV infection and serve as disseminators of the virus throughout the host (reviewed in reference 29). In immunocompetent hosts, primary CMV infections are generally asymptomatic, with immune cells either killing virus-infected cells or restricting viral cell-to-cell spread and repli-cation. The latter effect occurs via induction of an antiviral state in noninfected cells or the activation of immune cells by soluble mediators such as type I interferon (alpha/beta inter-feron [IFN-/]) and type II interferon (IFN-) (8, 10, 41, 50, 60, 73). Several hundred genes stimulated in response to both type I and type II IFNs have been identified by microarrays in various cell types over the years (18, 19, 37, 38, 66). In contrast, the recently discovered type III IFNs are not well characterized but may have comparable functions to the type I IFNs, mediated by shared downstream signaling and IFN-stimulated genes (ISGs) (3, 33, 42, 63, 72, 78, 84). Type III IFNs are