It has been amply documented that herpes simplex virus (HSV) persists in sensory ganglia of the peripheral nervous system (PNS). In contrast, HSV latency in the central nervous system (CNS) has not been well characterized. Corneal inoculation of virus results in a productive viral infection in the CNS during the first week after inoculation, indicating that the virus can progress from the PNS to the CNS. During latency, HSV has been found by co-cultivation of CNS tissue in only a very small fraction of inoculated mice. We have used here molecular hybridization techniques to analyse the fate of viruses that reach the CNS by anatomical pathways. We show that 6 days after corneal inoculation of HSV-1 a productive viral infection was present in brain tissue as well as in peripheral ganglia in at least 90%F of the inoculated mice. The mortality during this acute phase was only 2%. In the survivors, latent HSV could be recovered by explantation from 95% of the trigeminal ganglia, but only 5% of the brain tissue explants of the same mice yielded infectious virus. However, HSV DNA sequences were detected in the brains of 30% of mice which harboured latent HSV in their trigeminal ganglia. These results suggest that viruses that progress from the PNS into the CNS are not eliminated, but are capable of establishing a latent infection in the CNS that cannot be reactivated by explantation techniques.