In previous studies, the herpes simplex virus type 1 (HSV-1) mutant, in1814, which lacks the trans-inducing function of Vmw65, did not replicate in the trigeminal ganglia of mice following corneal inoculation but did establish a reactivatable latent infection in the ganglia 12 to 24 h after ocular infection. Since in1814 did not replicate in vivo, the molecular events during the establishment phase of latent HSV-1 infection could be characterized without the complications of concurrent productive viral infection. In comparison to parental HSV-1 strain 17+, the expression of viral immediate early (IE), early and late genes and the levels of viral DNA in the trigeminal ganglia of mice following in1814 infection were greatly reduced. However, accumulation of latency-associated transcripts, a prominent feature of latent HSV-1 infection, occurred in a wild-type fashion. Furthermore, low levels of viral gene expression and an increase in the level of viral DNA in the in1814-infected ganglia were not detected until 1 to 2 days after the establishment of HSV-1 latency. Thus, IE gene expression and replication of viral DNA in the trigeminal ganglia are not prerequisites for the establishment of HSV-1 latency. These results suggest that the pathways leading to productive and latent infections in neurons may diverge at an early stage of the host-HSV-1 interaction and that the level of viral IE gene expression has a key role in determining the outcome of infection.