A pseudorabies virus (PrV) mutant, deficient in the nonessential glycoprotein E (gE) and expressing the LacZ gene (gE- beta gal+ PrV), and its rescued virus were inoculated intranasally in mice. The median lethal dose of gE- beta gal+ PrV was similar to that of the parental Kaplan strain, but mice survived longer and did not develop symptoms of pseudorabies. In the nasal mucosa, gE- beta gal+ PrV replicated less efficiently than rescued virus. gE- beta gal+ PrV could infect first-order trigeminal and sympathetic neurons innervating the nasal mucosa. However, transneuronal transfer to second-order cells groups did not occur in trigeminal pathways and was severely reduced in sympathetic pathways. The mutant was also unable to propagate in the parasympathetic system. In contrast, gE-rescued virus was transferred transneuronally in trigeminal, sympathetic, and parasympathetic pathways, like wild-type PrV. These findings provide further evidence that deletion of gE specifically affects transneuronal transfer of PrV more than penetration and multiplication of the virus in first-order neurons.