The herpes simplex virus (HSV) gene RL1 encodes the protein ICP34.5, which is a specific neurovirulence factor. Null mutants in RL1 fail to replicate in the central nervous system of mice and are therefore totally non-neurovirulent. Additionally, they fail to replicate in neurons of the peripheral nervous system, although they are capable of establishing and reactivating from a latent infection. As the precise function of ICP34.5 in HSV-neuronal interactions is unknown, we have studied the role of ICP34.5 in vitro by examining in detail the phenotypes of RL1-negative viruses in two defined tissue culture systems. The first was mouse embryo fibroblast 3T6 cells, in which RL1-negative mutants are impaired and the in vivo phenotype is mimicked. This impairment is amplified when the cells are in the stationary state. The second was mouse embryo testicular carcinoma F9 cells which, in the undifferentiated state, provide a reversal of phenotype; wild-type virus fails to grow but RL1-negative virus replicates efficiently. Differentiation results in the ability to support wild-type virus growth. The stage at which the replication cycle is blocked plus the role of cellular factors is addressed in both tissue culture systems. Evidence is provided that cell type and cell state are crucial to ICP34.5-cellular interaction and hence, based on these parameters, ICP34.5 can be defined as a host-range determinant. Identification of cellular proteins that specifically interact with or are homologues of ICP34.5 may lead to the identification of neuron-specific proteins that have a similar role.