Murine peritoneal macrophages were rapidly rendered listericidal after exposure to lymphokine-rich supernatants (LRSs) derived from antigen-pulsed Listeria monocytogenes-immune spleen cells. A 6-h incubation period with LRSs was sufficient to induce microbicidal activity in resident macrophages. In vitro induction of macrophage listericidal activity by constant exposure to LRSs persisted for 18 h, after which time spleen cell factors were no longer capable of modifying intracellular inactivation of Listeria. Results obtained by utilizing a short assay indicated that the killing kinetics is extremely rapid, with large numbers of bacteria destroyed during the first 15 min of infection. Intracellular killing at this time appeared to be greatly dependent upon the stage of growth from which the microorganisms were harvested. Induction of bactericidal macrophages by infection of mice with a sublethal dose of virulent Listeria cells and subsequent intraperitoneal elicitation with heat-killed homologous bacteria was similarly a transient event. Macrophages harvested 18 h after antigenic challenge displayed dramatic antibacterial activity during the first 22 h in culture. After 22 h, activity was lost, and stasis was observed during the ensuing 23 h. At 68 h, macrophages were devoid of antilisterial action. Activity, however, could be recalled after incubation with LRSs.