A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar M r to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced M r (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in M r. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.