The present study used the rabbit iontophoresis model to examine the thymidine kinase (TK) requirement for HSV-1 latency, and test the round trip theory of latency with emergent phenotypic mutations of the HSV-1 TK negative (TK−) inoculating strain. The results demonstrated repeated induced ocular shedding of latent HSV-1 in 14–100% of rabbits. The TK phenotype of recovered tear film following spontaneous shedding and repeated iontophoresis induction wasthymidine Kinase negativein 90% of eyes. Sequential shedding of TK−virus from the same eye over time was demonstrated in 21% of eyes in 33% of rabbits. We conclude that TK isnotan absolute requirement for the establishment or reactivation of latent HSV-1 in the rabbit model. The round trip theory of latency wasnotsupported as TK+isolates and syncytial variants of the TK−inoculating strain which were recovered at the ocular surface after the initial iontophoresis couldnotbe demonstrated following subsequent trials of iontophoresis.