Studies using Listeria monocytogenes as an antitumor agent were initiated to determine the requirements for Listeria-mediated tumor inhibition to occur. When Strain 13 guinea pigs were injected with an admixture of viable Listeria and a methylcholanthrene-induced fibrosarcoma in a ratio of 1 bacterium to 100 tumor cells, Listeria had a marked capacity to inhibit tumor growth. This confirms an earlier study in our laboratory (M. M. Dustoor, A. Fulton, W. Croft, and A. A. Blazkovec, Infect. Immun. 23:54-60, 1979). At no time did animals exhibit overt symptoms of disease as a result of Listeria infection. Animals treated with antilymphocyte serum, which had previously been shown to abrogate T-cell functions, were no longer able to suppress Listeria-tumor cell mixtures. Treatment in vivo with carrageenan, a macrophage-inhibitory agent, also abrogated Listeria-mediated tumor inhibition. These results suggest that Listeria-mediated inhibition requires intact T-lymphocyte and macrophage function. Experiments in which Listeria was given in admixture with the tumor cells or in the opposite flank demonstrated that the antitumor effects require intimate association of the Listeria and tumor cells. Histopathological studies, showing that macrophages and lymphocytes are the predominant inflammatory cells present at sites of tumor destruction, further suggest a role for these cells in Listeria-mediated inhibition. Animals which had rejected prior Listeria-tumor cell inocula were resistant to rechallenge with the homologous tumor for more than 1 year. This work thus confirms in vitro studies demonstrating that both lymphocytes and macrophages are required for Listeria-mediated tumor inhibition to occur. This study demonstrates that viable Listeria can have potent antitumor effects without causing overt disease as a result of Listeria infection.