The biological activities of mycobacteria-reactive Lyt-2+ T cells were characterized in vitro. T cells from mice immunized with killed Mycobacterium tuberculosis or viable M. bovis were restimulated in vitro and cloned under limiting dilution conditions. Several L3T4-Lyt-2+ T cell lines, some of them KJ16+, were established. These T cell lines were capable of lysing mycobacteria-primed macrophages in an antigen-specific way. The cytolytic activity of some T cell lines was found to be class I restricted, whereas others showed antigen-specific killing in the absence of apparent H-2 restriction. Several T cell lines produced interferon-gamma after appropriate stimulation. Furthermore, these T cell lines could induce tuberculostatic macrophage capacities by apparently two different mechanisms, namely by secretion of lymphokines (most probably interferon-gamma) and by direct cell contact. We conclude that CD8 T cells with antigen-specific cytolytic potential are generated during tuberculosis and that these T cells are involved in the immune response to tubercle bacilli.