Two herpes simplex virus (HSV) intertypic recombinants were isolated with genomes composed entirely of HSV-2(186) nucleotide sequences except for a 6.0 Kb segment of HSV-1(17) DNA positioned between 0.40 and 0.44 map units. Following corneal infection of mice, HSV-1(17) and the two intertypic recombinants spread from infected eyes into the central nervous system and induced a fatal encephalitis. Ocular infection with the HSV-2 (186) parent did not lead to detectable amounts of virus in the brain, and none of the mice developed encephalitis. The 6.0 Kb HSV-1(17) DNA inserted within the genome of the two intertypic recombinants contained nucleotide sequences involved in DNA replication. These include the HSV-1(17)oriL, the HSV-1(17) gene for DNA polymerase and portions of the HSV-1(17) gene coding for DNA-binding protein ICP8. Thus, our results indicate that the difference in the capacity of HSV-1(17) and HSV-2(186) to spread from the cornea into the CNS is determined solely by nucleotide sequences associated with DNA replication.