Insertional mutagenesis was used to construct an equine herpesvirus 1 (EHV-1) mutant in which the open reading frame for glycoprotein D was replaced by a lacZ cassette. This gD deletion mutant (delta gD EHV-1) was unable to infect normally permissive RK cells in culture, but could be propagated in an EHV-1 gD-expressing cell line (RK/gD). Phenotypically complemented delta gD EHV-1 was able to infect RK cells, but did not spread to form syncytial plaques as seen with wild type EHV-1 or with delta gD EHV-1 infection of RK/gD cell cultures. Therefore EHV-1 gD is required for virus entry and for cell-cell fusion. The phenotypically complemented delta gD EHV-1 had very low pathogenicity in a mouse model of EHV-1 respiratory disease, compared to a fully replication-competent EHV-1 reporter virus (lacZ62/63 EHV-1). Intranasal or intramuscular inoculation of mice with delta gD EHV-1 induced protective immune responses that were similar to those elicited in mice inoculated with lacZ62/63 EHV-1 and greater than those following inoculation with UV-inactivated virus.