We compared the abilities of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and sensitized murine lymph node lymphocytes to activate syngeneic murine peritoneal macrophages to inhibit the growth of intracellular Mycobacterium bovis BCG in vitro. IFN-gamma could activate antimycobacterial defense only when added to macrophage cultures prior to their infection with BCG. TNF-alpha was without any effect. In contrast, BCG-sensitized lymphocytes could induce antimycobacterial defenses when added after macrophages had been infected with BCG. The cell-mediated effect required direct contact between effector lymphocytes and the targets (BCG-infected macrophages), as revealed in studies in which these cell populations were separated by a semipermeable membrane. Cyclosporin A, which inhibits the production of relevant macrophage-activating lymphokines, did not abrogate the ability of sensitized lymphocytes to activate antimycobacterial effects in infected macrophages. Furthermore, only BCG-sensitized lymphocytes, and not Listeria-sensitized lymphocytes, could activate the antimycobacterial effects. These lymphocytes were not cytotoxic to the infected macrophages. The presence of anti-TNF-alpha antibody in cocultures reduced the antimicrobial effects. We propose that the activation of antimycobacterial defense in macrophages can occur by direct physical contact with sensitized lymphocytes. This process may be due to lymphocyte membrane-associated TNF-alpha, as we previously demonstrated in our studies of antileishmanial defense.