To develop a live vaccine for equine herpesvirus type 1 (EHV-1), two EHV-1 mutants containing no heterogeneous DNA, ΔgI and ΔgE, were constructed with deletions in the open reading frame of either glycoprotein I (gI) or E (gE), respectively. In equine cell culture, deletion mutants formed smaller plaques than the parental and revertant viruses, but the one-step growth patterns of the deletion mutants and the parental strain were approximately the same. These results suggest that both gI and gE contribute to the ability of EHV-1 to spread directly from cell-to-cell, but that these glycoproteins are not required for viral growth in vitro. Mice and hamsters inoculated intranasally with these mutants showed no clinical signs, and continued to gain weight, whereas those inoculated with the parental virus exhibited a reduction in mean body weight. Furthermore, nervous manifestations were observed in hamsters inoculated with the parental virus. These results suggest that gI and gE have an important role in EHV-1 virulence including neurovirulence in experimental animal models. On the other hand, serum neutralizing antibodies were detected in mice immunized with ΔgI or ΔgE at two weeks after inoculation. Following challenge with the parental virus, ΔgI- or ΔgE-immunized mice were able to clear parental virus from their lungs faster than mock-immunized mice. These results suggest that the EHV-1 mutants defective in gI and in gE are attenuated but have ability to elicit immune responses in inoculated mice that contribute to virus clearance.