The UL24 gene of herpes simplex virus overlaps the viral thymidine kinase (tk) gene. Most previous studies of UL24 have examined UL24 mutants that have also contained tk and sometimes other mutations. To address the importance of UL24 for viral replication in cell culture and in infections of a mammalian host, we constructed a mutant virus containing a UL24 nonsense mutation that does not affect TK activity and a second mutant that contains clustered point mutations in UL24 and a mutation in tk that does not by itself affect the ability of the virus to replicate acutely in mouse ganglia or to reactivate from latent infection following corneal inoculation of mice. Both mutant viruses replicated in cells in culture and in the mouse eye, albeit less efficiently than wild type or control viruses. Both mutants were much more severely impaired for acute replication in trigeminal ganglia and for reactivation from latency following explant of these ganglia. Viral DNA and latency-associated transcripts were present, albeit at lower levels in ganglia infected with the nonsense mutant. These results indicate that UL24 is especially important for productive infection of mouse sensory ganglia and may have implications for the behaviors of certain tk mutants in pathogenesis.