Almost all mammalian alphaherpesviruses can grow in cells derived from several types of animals in vitro. However, FHV-1 can only infect feline cell lines. For this reason, FHV-1 should be a good model to investigate species barriers to herpesviruses in vivo. To apply bacterial mutagenesis of FHV-1, we cloned the FHV-1 genome as a BAC. Using lambda and flp recombinations, we introduced a monomeric red fluorescence protein into the C-terminus of glycoprotein D. Although GFP in the constructed recombinant FHV-1, a transfectant of the bacmid of FHV-1 that possessed the GFP, acted in non-feline cell lines, the virus could not enter non-feline cell lines, demonstrating that the host specificity of FHV-1 was restricted in an early step of infection. The host range of canine herpesvirus is limited to dogs in vitro and in vivo; it cannot enter non-canine cell lines as a result of infection but the GFP is active by transfection, revealing the same result that the restriction step is at an early stage of infection. These results suggest the possibility of breaking species barriers of FHV-1 and CHV by modifying the gene(s) that act at the early stage of infection.