Ten monoclonal antibodies (MAbs) were produced against equine herpes virus type 1 (EHV1). Two appeared type-specific, while the other eight were directed against epitopes common to both EHV1 and EHV4. Two MAbs directed against the glycoprotein gp2 recognized linear epitopes, as demonstrated by Western blotting. With pools of type-specific MAbs, 282 field isolates were typed in an immunoperoxidase monolayer assay (IPMA). From a total of 254 fetal or neonatal isolates, 244 (96%) were typed as EHV1, whereas 14 out of 15 (93%) respiratory tract isolates were typed as EHV4. Surprisingly, 3 out of 13 isolates (23%) originating from horses with neurological disease were typed as EHV4. No antigenic differences were found among 75 randomly selected EHV1 field isolates, using the panel of ten MAbs and six additional MAbs, directed against gp2, gB, or gC. Typing by restriction endonuclease analysis with BamHI corresponded completely with that of MAb analysis. There was a remarkable degree of uniformity in BamHI restriction patterns, with 90% of the investigated EHV1 isolates belonging to the 1P electropherotype. Among 30 randomly selected EHV1 isolates we could not identify the EHV1.1B electropherotype, which has been the predominant electropherotype in Kentucky since 1982. Mobility differences were seen in fragments originating from the repeat regions. These differences were not caused by heterologous cell passage, since all viruses were passaged in equine cell systems.