The cell culture-adapted KyA strain of equine herpesvirus type 1 (EHV-1) has been found to be attenuated in young horses (Matsumura et al., 1996, Vet. Microbiol. 48, 353-365). The KyA strain lacks at least six genes in its genome, including those encoding glycoproteins gE and gI. To elucidate whether EHV-1 glycoproteins gE and gI play a role in viral virulence, we have constructed an EHV-1 recombinant that has the genes encoding both gE and gI deleted from its genome and its revertant. Growth properties of the deletion mutant virus in vitro were compared with those of the parent and the revertant viruses. Plaque size of the mutant virus in fetal horse kidney (FHK) cells was significantly smaller than those of the parent and the revertant viruses. In one-step growth experiments, however, the yields of infectious virus from FHK cells infected with the deletion mutant, the parent, or the revertant virus were approximately the same. The results suggested that gE and/or gI of EHV-1 promoted cell-to-cell spread of the virus, but that these glycoproteins were not involved in the process of virus maturation and release or in virus attachment and penetration. Subsequently, the virulence of mutant and revertant viruses was examined in young horses. No clinical signs were observed in six horses, including three colostrum-deprived foals inoculated intranasally with the deletion mutant virus, whereas three colostrum-deprived foals inoculated intranasally with the revertant virus manifested clinical signs typical for EHV-1 respiratory infection (i.e., pyrexia, nasal discharge, and swelling of submandibular lymph nodes). The results obtained from in vivo studies revealed that the EHV-1 mutant defective in both gE and gI genes was avirulent in young horses, suggesting that gE and/or gI of the EHV-1 have an important role in EHV-1 virulence. However, the EHV-1 mutant defective in both gE and gI genes induced only a partial protectivity in inoculated foals from manifestation of respiratory symptoms after challenge infection.