To determine the effect of interferon (IFN) on the pathogenesis of pseudorabies virus (PRV), porcine nasal mucosal explants were first treated with recombinant porcine methionyl-IFN-alpha 1 and then infected with one of three strains of PRV. The stroma of treated mucosal explants were protected against infection with virulent PRV or PRV of intermediate virulence because the infection was restricted to the epithelial cells. In contrast, untreated mucosal explants were readily infected by virulent PRV or PRV of intermediate virulence; the infection spread from epithelial cells to stromal fibroblasts. Avirulent PRV infection was restricted to the epithelial cells of treated and untreated mucosal explants. IFN treatment limited the extent of all three PRV infections in the epithelial cells. Cultured porcine fibroblasts and porcine kidney cells were also treated with IFN and subsequently infected with PRV; infection was prevented in most porcine fibroblasts and the virus yield from porcine kidney cells was reduced. Budding of virulent PRV nucleocapsids through the inner nuclear membrane was observed more frequently in treated than in untreated mucosal explants were enveloped virus particles accumulated between the inner and outer nuclear membranes, indicating that the membrane-associated events of PRV replication had been affected. We conclude that IFN-alpha protects stromal fibroblasts against PRV infection and reduces virus replication in epithelial cells.