We previously described that natural autocytolytic cells (NACs) which were detected in the mesenteric lymph nodes (MLNs) of normal mice, did lyse leukocytes contained in the same lymph nodes. Discrete plaques were observed in monolayers of leukocytes prepared in wells of a microculture plate by NACs phagocytizing and lysing other leukocytes. A large NAC which was morphologically similar to macrophage was found in the middle of each plaque. The mechanisms of autocytolysis by NACs were further investigated in the present study. In contrast to various types of autocytotoxic cells reported previously, for which the incubation with fetal calf serum (FCS) was a prerequisite, the NACs showed their cytolytic activity even in serum-free medium. This excluded the possible "sensitization" by FCS proteins in vitro. The presence of the phagocytosis inhibitor cytochalasin B eliminated the autocytolysis. The peroxide scavenger thioglycollate broth had no effect on the formation of autocytolytic plaques. These results further support the previous finding that autocytolytic plaques were formed by NACs phagocytizing other leukocytes. Autologous nonadherent spleen cells and erythrocytes were also phagocytized by MLN-NACs with plastic adherent characteristics. Ia antigen was demonstrated on the surface of an approximately half population of NACs but neither Thy-1.2 antigen nor immunoglobulins. NACs may represent a subpopulation of macrophages, although the majority of adherent, phagocytic macrophages did not show the NAC activity. The endotoxin lipopolysaccharide (LPS) of Escherichia coli was apparently able to activate MLN-NACs of BALB/c mice in vivo when injected intraperitoneally at a dose of 10 micrograms/ml. A peak of the NAC activity was observed 3 days after the injection.(ABSTRACT TRUNCATED AT 250 WORDS)