The ability of alpha interferon (IFN-α) and IFN-γ to inhibit transmission of herpes simplex virus type 1 (HSV-1) from neuronal axon to epidermal cells (ECs), and subsequent spread in these cells was investigated in an in vitro dual-chamber model consisting of human fetal dorsal root ganglia (DRG) innervating autologous skin explants and compared with direct HSV-1 infection of epidermal explants. After axonal transmission from HSV-1-infected DRG neurons, both the number and size of viral cytopathic plaques in ECs was significantly reduced by addition of recombinant IFN-γ and IFN-α to ECs in the outer chamber in a concentration-dependent fashion. Inhibition was maximal when IFNs were added at the same time as the DRG were infected with HSV-1. The mean numbers of plaques were reduced by 52% by IFN-α, 36% by IFN-γ, and by 62% when IFN-α and IFN-γ were combined, and the mean plaque size was reduced by 64, 43, and 72%, respectively. Similar but less-inhibitory effects of both IFNs were observed after direct infection of EC explants, being maximal when IFNs were added simultaneously or 6 h before HSV-1 infection. These results show that both IFN-α and IFN-γ can interfere with HSV-1 infection after axonal transmission and subsequent spread of HSV-1 in ECs by a direct antiviral effect. Therefore, both IFN-α and -γ could contribute to the control of HSV-1 spread and shedding in a similar fashion in recurrent herpetic lesions.