Two herpes simplex virus (HSV) glycoproteins E and I (gE and gI) form a heterooligomer which acts as an Fc receptor and also facilitates cell-to-cell spread of virus in epithelial tissues and between certain cultured cells. By contrast, gE-gI is not required for infection of cells by extracellular virus. HSV glycoproteins gD and gJ are encoded by neighboring genes, and gD is required for both virus entry into cells and cell-to-cell spread, whereas gJ has not been shown to influence these processes. Since HSV infects neurons and apparently spreads across synaptic junctions, it was of interest to determine whether gD, gE, gI and gJ are also important for interneuronal transfer of virus. We tested the roles of these glycoproteins in neuron-to-neuron transmission of HSV type 1 (HSV-1) by injecting mutant viruses unable to express these glycoproteins into the vitreous body of the rat eye. The spread of virus infection was measured in neuron-rich layers of the retina and in the major retinorecipient areas of the brain. Wild-type HSV-1 and a gJ- mutant spread rapidly between synaptically linked retinal neurons and efficiently infected major retinorecipient areas of the brain. gD mutants, derived from complementing cells, infected only a few neurons and did not spread in the retina or brain. Mutants unable to express gE or gI were markedly restricted in their ability to spread within the retina, produced 10-fold-less virus in the retina, and spread inefficiently to the brain. Furthermore, when compared with wild-type HSV-1, gE- and gI- mutants spread inefficiently from cell to cell in cultures of neurons derived from rat trigeminal ganglia. Together, our results suggest that the gE-gI heterooligomer is required for efficient neuron-to-neuron transmission through synaptically linked neuronal pathways.