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Pseudorabies virus (PRV) has been shown to be an effective transneuronal tracer within both the peripheral and the central nervous system. The only investigations of this virus in the visual system have examined anterograde transport of PRV from injection sites in the retina. In the present study, we injected attenuated forms of PRV into the primary visual cortex of both rats and cats to determine whether transneuronal retrograde infection would occur back to the retina. In rats, we made small injections into visual cortex of a strain of PRV (Bartha Blu) that contained a beta-galactosidase promoter insert. In cats, we injected PRV-M201 into area V1 of visual cortex. After a 2- to 4-day incubation period, we examined tissue from these animals for the presence of the beta-galactosidase marker (rats) or the virus itself (cats). Cortical PRV injections resulted in transneuronal retrograde infection of the lateral geniculate nucleus (LGN), thalamic reticular nucleus (TRN), and retina. PRV was retinotopically distributed in the pathway. In addition, double-labeling experiments in cats using an antibody against gamma-aminobutyric acid (GABA) were conducted to reveal PRV-labeled interneurons within the LGN and TRN. All TRN neurons were GABA+, as was a subset of LGN neurons. Only the subset of TRN neurons adjacent to the PRV-labeled sector of LGN was labeled with PRV. In addition, a subset of GABA+ interneurons in LGN was also labeled with PRV. We processed some tissue for electron microscopy to examine the morphology of the virus at various replication stages. No mature virions were detected in terminals from efferent pathways, although forms consistent with retrograde infection were encountered. We conclude that the PRV strains we have used produce a local infection that progresses primarily in the retrograde direction in the central visual pathways. The infection is transneuronal and viral replication maintains the intensity of the label throughout the chain of connected neurons, providing a means of examining detailed circuitry within the visual pathway.