Background Newly delivered herpes simplex virus genomes are subject to repression during the early stages of infection of human fibroblasts. This host defence strategy can limit virus replication and lead to long-term persistence of quiescent viral genomes. The viral immediate-early protein ICP0 acts to negate this negative regulation, thereby facilitating the onset of the viral replication cycle. Although few mechanistic details are available, the host repression machinery has been proposed to assemble the viral genome into a globally inaccessible configuration analogous to heterochromatin, blocking access to most or all trans-acting factors. The strongest evidence for this hypothesis is that ICP0-deficient virus is unable to reactivate quiescent viral genomes, despite its ability to undergo productive infection given a sufficiently high multiplicity of infection. However, recent studies have shown that quiescent infection induces a potent antiviral state, and that ICP0 plays a key role in disarming such host antiviral responses. These findings raise the possibility that cells containing quiescent viral genomes may be refractory to superinfection by ICP0-deficient virus, potentially providing an alternative explanation for the inability of such viruses to trigger reactivation. We therefore asked if ICP0-deficient virus is capable of replicating in cells that contain quiescent viral genomes. Results We found that ICP0-deficient herpes simplex virus is able to infect quiescently infected cells, leading to expression and replication of the superinfecting viral genome. Despite this productive infection, the resident quiescent viral genome was neither expressed nor replicated, unless ICP0 was provided in trans. Conclusion These data document that quiescent HSV genomes fail to respond to the virally modified host transcriptional apparatus or viral DNA replication machinery provided in trans by productive HSV infection in the absence of ICP0. These results point to global repression as the basis for HSV genome quiescence, and indicate that ICP0 induces reactivation by overcoming this global barrier to the access of trans-acting factors.