The cell-mediated immune (CMI) response of foals to virulent equine herpesvirus-1 (EHV-1) infection was evaluated by multi-parameter flow cytometry (FCM). Ten 7-8-month-old EHV-1 seronegative foals were infected intranasally with virulent EHV-1 and 10 foals served as uninfected controls. Blood samples were collected 6 and 7 weeks after infection to test for specific CMI responses to live heterologous EHV-1 recall antigen. The activation markers included major histocompatibility complex class II (MHC II), intracellular interferon gamma (IFN-gamma) and interleukin 4 (IL-4). The results from both tests were averaged before statistical analysis. Following EHV-1 stimulation, the MHC II expression index (EI) increased significantly in CD2+CD4+CD8- and CD2+CD4-CD8+ subsets of the infected group. At 4 days after incubation, the non-antigen stimulated CD2+CD4-CD8- subset of the infected group expressed a high percentage (61.1%) of MHC II. When stimulated with EHV-1, the MHC II expression declined significantly but remained at a relatively high percentage (34.4%). The IFN-gamma EI was significantly higher in infected foals in all major T cell subsets (CD2+) while only the CD2+CD4+CD8- subset showed a significant increase in intracellular IL-4 EI. The FCM results showed strong specific CMI responses to EHV-1 by all three tested parameters compared to the control group (p<0.01). The high MHC II expression in the CD2+CD4-CD8- subset suggests that this T cell subset may represent a gammadelta TCR repertoire and thereby plays an important role as antigen presenting cells in the horse, as reported in other species. Being able to simultaneously quantify the frequency of specific lymphocyte subsets and the expression of cytokines that characterize activation of lymphocytes and protective CMI by multi-parameter FCM enables evaluation of subset-specific CMI responses to EHV-1 infection. This system can be applied to measure CMI responses to other equine vaccines and pathogens.