Previous studies in the mouse and guinea pig have reported little or no colonization of sensory ganglia by strains of herpes simplex type 1 failing to express the enzyme, thymidine kinase (TK). The current study in the rabbit demonstrated trigeminal ganglionic colonization and reactivation of a latent thymidine kinase negative strain of HSV-1 by two independent methods: iontophoresis-induced ocular shedding and co-cultivation. Treatment with topical steroids during the acute infection did not enhance the latency rate. Following reactivation, back mutation with phenotypic reversion to thymidine kinase positive was demonstrated in a few recovered isolates. The current study also emphasized the importance of species differences to explain differing experimental results in studies of HSV-1 TK negative latency.