The relative contributions and specific functions of T cell subsets in cattle infected with Mycobacterium avium ss paratuberculosis are not well known. To help elucidate their roles, T-lymphocyte subsets were purified using commercially available monoclonal antibodies to CD3, CD4, CD8, and gamma-delta lymphocyte cell surface markers, and a magnetic bead separation column system. Purity of subsets was verified by flow cytometry. Subsets were assayed for proliferation and interferon-gamma (IFN-gamma) production in response to phytohemagglutinin (PHA) and M. avium ss paratuberculosis antigens (APPD). IFN-gamma was measured by antibody-capture ELISA, and OD values converted to picograms of IFN-gamma using a standard curve established with recombinant bovine IFN-gamma. The results indicated correlation between proliferation responses and IFN-gamma production in response to specific antigens, but not PHA. CD4+ lymphocytes were the most prolific producers of IFN-gamma. CD8+ lymphocytes produced IFN-gamma to a lesser extent, whereas gamma-delta T lymphocytes produced little or no IFN-gamma. There was no significant difference in the levels of IFN-gamma produced by whole PBMC and CD4+ vs CD8+ cells, and CD4+ vs gamma-delta+ cells in response to PHA were significant at P<0.01, but there was no significant difference between CD8+ and gamma-delta + cell IFN-gamma responses. In response to A-PPD, the differences between CD4+ and gamma-delta + cells was significant at P<0.05, but levels of IFN-gamma produced to specific stimulation were generally lower. These data for cattle are similar to observations made in other animal species, where CD4+ cells are the major type of T lymphocytes producing IFN-gamma. They further suggest that whatever the role gamma-delta T cells play in paratuberculosis it is not likely to be mediated by IFN-gamma production.