A latent state of the herpes simplex virus type 2 genome was established in a human neuroblastoma cell line (SMS-KCNR) to initiate studies on the mechanism by which host cells interact and regulate latent viral genes. To establish viral latency, it was necessary to prevent virus replication by briefly exposing the infected cells to antiherpetic acycloguanosine (20 microM) and human interferon (120 U/ml). Subsequently however, these cells could be propagated without any antiherpetic agents and almost 60% of the cell population contained viral genome. While these cells did not produce any infectious virus, immunoblot analysis revealed two intracellular polypeptides with molecular weights of 87.5 kDa and 67 kDa, respectively, that interacted with hyperimmune anti-HSV2 rabbit serum. Two cellular enzymes, acetylcholinesterase and choline acetyltransferase, involved in metabolism of neurotransmitters were expressed at a higher level in the latently infected cells than in the mock-infected control cells. Infectious HSV-2 could be reactivated from these cells only after the cells had undergone massive morphological differentiation and maturation to flat cell types by extensive treatment with 20 micron bromodeoxyuridine.