Abstract A panel of six neutralizing monoclonal antibodies (MAbs), neutralization-resistant variant (escape mutant [EM]) viruses, and individual viral proteins derived from a vaccinia virus expression system were used to identify the neutralizing determinants of equine arteritis virus (EAV). The neutralizing MAbs recognize a single neutralization site on the 29-kDa envelope glycoprotein of EAV (U. B. R. Balasuriya et al., 1993, J. Gen. Virol., 74, 2525-2529). Vaccinia virus recombinants which express either the G L protein or the M protein of EAV, and a G L-specific antipeptide serum were used to prove that the 29-kDa glycoprotein recognized by the MAbs is the G L protein. The MAbs were used to select a panel of seven EM viruses, whose phenotypic properties were characterized by neutralization and Western immunoblotting assays. The neutralizing MAbs segregated into three groups on the basis of these assays, indicating that they define three interactive epitopes on the G L protein. Sequencing of the entire open reading frame (ORF) 5, which encodes the G L protein, from each EM virus identified the nucleotide mutations responsible for the altered phenotypic properties exhibited by the EM viruses. Compared to the sequence of ORF 5 of the parent strain (EAV-UCD), all nucleotide changes occurred within a span of 17 nucleotides (11423 to 11439). Phenotypic alterations in the EM viruses probably were the result of amino acid substitutions within a region of six amino acids (99 to 104), all of which focally altered the predicted hydrophobicity and/or secondary structure of the G L protein. We conclude that this region constitutes an important neutralization domain of EAV.