Alveolar macrophages (AM) from lungs of normal F344 rats can be rendered tumoricidal by incubation in vitro with either muramyl dipeptide (MDP) at a minimum dose of 10 micrograms/ml or undiluted cell-free culture supernatants from mitogen-stimulated F344 rat lymphocytes rich in macrophage-activating factor (MAF) activity. Neither MAF at dilutions exceeding 1:6 nor MDP at doses lower than 10 micrograms/ml activated AM to become tumor cytotoxic. The combination of agents at subthreshold amounts (MAF 1:18; MDP 0.001 to 1 microgram/ml) activated AM to significant levels of cytotoxicity. AM activated by these agents were rendered tumoricidal and destroyed syngeneic, allogeneic, and xenogeneic tumor targets in vitro. The synergism for AM activation between preparations of MAF and MDP required that AM be incubated first with MAF and then with MDP. Even a 15-min treatment of AM with MAF conditioned the cells to respond to subthreshold amounts of MDP and to be rendered tumoricidal. Since treatment of MAF and MDP with polymyxin B did not interfere with macrophage activation, we were able to rule out the possibility that our preparations were contaminated with lipopolysaccharide. Synergism for AM activation was demonstrated also when AM were treated with MAF and MDP encapsulated within liposomes. This finding suggests that the binding of agents to the macrophage surface is not a prerequisite for the synergistic activation of AM by MAP and MDP.