Using a blastogenesis assay of peripheral blood mononuclear cells depleted of specific lymphocyte subsets, various subset add-back experiments, and examination of T cell lines from an animal immunized with M. paratuberculosis, we examined the functional activity of T cell subsets in response to infection. Initial response to M. paratuberculosis antigens was mediated by BoCD4+ T helper lymphocytes reacting with cell-wall antigens during the first 2-3 months. Cellular concentrations of BoCD4+ lymphocytes then rapidly declined and anergy developed. Responsiveness during this 70 day period could not be restored. A second proliferative phase was shown to be mediated by BoCD8+ and N cells reacting to soluble antigens of M. paratuberculosis. N and CD8+ cell populations accounted for the total proliferative response of peripheral blood cells. By a series of add-back experiments and examination of N2+ cell lines, it was concluded that antigen-specific BoCD8+ and N populations respond to M. paratuberculosis antigens by a BoCD4+-independent mechanism. Proliferation of BoCD8+ and N cells were also independent of each other. The BoCD8+ cell population exerted a moderate suppressive effect on both specific and nonspecific responses, but no antigen-specific suppression by the BoCD8+ population could be demonstrated. The N population, however, exhibited an antigen-specific suppression of BoCD4+ cells. Add-back experiments illustrated that the BoCD4+ population contained M. paratuberculosis-specific reactive cells which failed to proliferate as a result of an antigen-specific suppression by the N population. It was concluded that the inability of effector cell populations to prevent intracellular proliferation of M. paratuberculosis was due to suppression of the T helper lymphocyte population required for macrophage activation.