Primary inoculation of mice and rabbits with an avirulent HSV-1 thymidine kinase negative (TK−) mutant reduced keratitis, mortality, and superinfection of the trigeminal ganglion (TG) as measured by cocultivation and iontophoresis-induced ocular shedding following ocular challenge with HSV-1 McKrae and W strains. However species differences were demonstrated; with complete protection in rabbits, and incomplete protection in mice. In mice, BUDR/autoradiography and restriction enzyme analysis identified both HSV-1 McKrae and W strains as having super infected the TG, established latency and reactivated. Also, the avirulent TK negative inoculating strain was altered to a virulent TK positive strain through possiblein vivoselection, mutation&/or host-modification, and possiblein vivorecombination with the virulent challenge strains. We conclude that lower species differences require that potential HSV-1 vaccines be tested in non-human primates prior to clinical trials, and that a DNA-free subunit herpes vaccine represents a safer alternative to a live virus vaccine.