Intracutaneous immunization of mice with 10(5) or 10(6) viable listeria resulted in acquired cellular resistance (ACR) of short duration (7 days). The period during which viable Listeria monocytogenes had to be present in order to induce ACR was estimated by killing the listeria at different times after immunization by injecting the bactericidal antibiotic amoxycillin. The killing of listeria within 6 h after injection prevented the induction of A CR completely, between 6 and 12 h partially, while survival of listeria within animals for at least 18 h was required for the induction of complete protection. To determine whether multiplication of viable listeria was a prerequisite for the induction of ACR, the bacteriostatic antibiotic minocycline was injected for four days after immunization. Induction of ACR was only possible if the dose of viable listeria was large enough to permit a proportion of the listeria to escape bacteriostasis. Interaction of peritoneal macrophages of normal mice and viable listeria yielded a supernatant which induced specific ACR in normal recipient mice. No ACR could be induced with supernatant obtained from normal macrophages after digestion of killed listeria. A reduced level of ACR was obtained with supernatant collected after interaction of macrophages from immune mice and viable listeria. The immunogenic material present in the supernatant of normal macrophages after interaction with viable listeria is thermolabile, has a molecular weight of over 300,000, and is not affected by treatment with DNase, RNase, or trypsin.