Macrophages from endotoxin (LPS)-unresponsive C3H/HeJ mice fail to develop tumoricidal activity after any of several in vivo or in vitro treatments that induce cytotoxicity with cells from LPS-responsive C3H/HeN mice. Under certain conditions, however, these macrophages could become tumoricidal: macrophages from in vivo immune reactions such as those induced by BCG infection, but not cells from irritant-induced peritoneal exudates, developed full cytotoxic capability after further exposure in vitro to microgram per milliliter concentrations of LPS or to supernatants from antigen-stimulated leukocyte cultures (lymphokines). Capacity of macrophages from BCG-infected C3H/HeJ mice to become tumoricidal after LPS exposure was gradually lost with time in culture and was absent by 24 hr. Requirements for at least two activation stimuli for cytotoxic activity with C3H/HeJ macrophages could also be fulfilled entirely in vitro: cells cultured in lymphokines plus LPS were fully tumoricidal; macrophages cultured in either agent alone were not cytotoxic. Similar interactions between lymphokines and LPS could be demonstrated with macrophages from LPS-responsive C3H/HeN mice. Tumoricidal activity by C3H/HeN macrophages cultured in lymphokines plus nanogram per milliliter concentrations of LPS was considerably greater than that by cells cultured in either agent alone. LPS and lymphokines were synergistic in their action on macrophage cytotoxicity. The synergistic effect of LPS on cytotoxicity by lymphokine-activated macrophages was evident after a 15-min pulse and was not dependent upon fluid-phase LPS. Synergistic action, however, was dependent upon 1.) treatment sequence: LPS was active only if given simultaneously with or after lymphokine treatment; LPS exposure before lymphokine treatment was ineffective, and 2.) treatment interval: capacity of lymphokine-treated macrophages to express cytotoxic activity after LPS exposure decayed with time in culture and was lost by 24 hr. Thus, macrophage activation for tumor cytotoxicity in both C3H/HeJ and C3H/HeN mice was the final result of a cascade of short-lived intermediary reactions in a defined sequence. Tumoricidal activity of fully activated cells and responsiveness of each noncytotoxic precursor cell to activation stimuli were all short lived macrophage reactions. Present evidence suggests that none of these reactions was reversible. Completion of each state of macrophage activation was completely dependent upon the simultaneous presence of localized activation signals and macrophage precursors able to respond.