Johne's Disease, a chronic inflammatory intestinal disease of ruminants, is caused by M. subsp avium paratuberculosis (Map). Available diagnostic assays including Map antigen ELISAs and IFN-γ assays are not sufficiently sensitive to identify animals in the early stages of disease. In the present study, we show that infected animals can be detected as early as 6 months following infection, using flow cytometry and monoclonal antibodies to phenotype T cells responding to Map antigens in vitro. Calves were experimentally exposed to Map per os at the time of birth and examined monthly for appearance of an immune response to Map (n = 5). A consistent proliferative response to PPD and soluble Map antigens was detected 6 months after exposure. CD4⁺/CD45R0⁺ memory T cells expressing activation molecules CD25, CD26, and ACT1 were the primary cell type responding to the antigens. Although CD8⁺ memory T cells proliferated in response to the antigens, they comprised a small proportion of responding cells. γδ T cells from infected and uninfected control animals (N = 3) proliferated in the presence of antigen. Natural Killer cells (NK) proliferated to a varying extent in cultures with and without antigen. The infected calves remained positive in the flow cytometric assay for the duration of the study (26 months). Control calves remained negative. With the use of Map specific antigens it should now be possible to more reliably identify animals in the early stages of infection using in vitro culture and flow cytometry.