A recombinant HSV-1 virus expressing EGFP from the HCMV major immediate early promoter (KOS-CMVGFP) was constructed to monitor viral replication and spread in vitro and in mice. KOS-CMVGFP replicated as efficiently as wild-type virus, strain KOS, in single cycle growth experiments in Vero cells indicating that the recombinant virus has no significant growth defects in vitro. Following ocular inoculation of mice, KOS-CMVGFP exhibited slight but statistically significant reductions in mouse tear film titers relative to wild-type virus. Progression of virus infection of the eyes, periocular tissue, and snout was readily followed by fluorescence microscopy. Insertion of the EGFP expression cassette into the KOS genome had no effect on the efficiency of establishment of latency as determined by quantitative competitive PCR of viral genomes in latently infected TG. KOS-CMVGFP reactivated with wild-type kinetics and efficiency by explant cocultivation, but exhibited a significant delay in the kinetics and a modest reduction in the efficiency of reactivation compared to KOS in the more sensitive TG cell culture model. Notably, EGFP expression preceded the detection of infectious virus by greater than 24 h in both ex vivo models and thus is a useful marker of the early stages in the induction of reactivation.