Specific serological diagnosis of equine herpesvirus 4 (EHV4; equine rhinopneumonitis virus) and EHV1 (equine abortion virus) hitherto has not been possible because of extensive antigenic cross-reactivity between these two closely related but distinct viruses. Recently, we identified EHV4 glycoprotein G (gG) and characterized it as a type-specific, secreted glycoprotein (B. S. Crabb, H. S. Nagesha, and M. J. Studdert, Virology 190:143-154, 1992). This paper shows that EHV1 gG also possesses type-specific epitopes and describes the localization of strong, type-specific epitopes to the apparently corresponding and highly variable regions comprising amino acids 287 to 382 of EHV4 gG and 288 to 350 of EHV1 gG. Fusion proteins expressing these variable regions reacted strongly and type specifically with sera from four foals, three of which were colostrum-deprived, specific-pathogen-free foals, whose history with respect to exposure to EHV4 or EHV1 was well-defined. These antigens provided the basis for the development of a single-well diagnostic enzyme-linked immunosorbent assay to distinguish horses infected with EHV4, EHV1, or both. Such a type-specific test provides for the first time the opportunity to differentiate antibodies to these viruses, and it has, therefore, important implications for understanding the epidemiology of these equine pathogens. Evidence for the existence of EHV1 in Australia 10 years prior to the first confirmed case of EHV1 abortion is presented.