Previous studies have shown that peritoneal macrophages from mice chronically infected with Bacillus Calmette-Guérin (BCG) become highly cytotoxic for tumor targets upon further in vitro triggering with a variety of agents. In the current studies, achievement of this activated state was characterized by the production and release of a cytotoxin, herein termed cytolytic factor (CF), which appeared in the fluid phase. Production/release of CF by the macrophage required transcription, translation, glycosylation, and an intact secretory apparatus, as evident from inhibition by treatment with actinomycin-D, cycloheximide, tunicamycin, and monensin, respectively, prior to and during triggering with lipopolysaccharide (LPS). CF obtained by culture of BCG-activated macrophages appeared rapidly in the supernatant after triggering. Using the actinomycin-D-treated L-929 or EMT-6 targets in a microassay, CF secreted by macrophages cultured in low molecular weight serum components was detected as an approximately 150-kD component on Sephacryl S-200. CF demonstrated a spectrum of cytotoxic activity against a number of tumor and normal targets in vitro. Its lytic activity appeared equally effective whether the targets were cultured in medium containing fetal calf serum (FCS) or in Neuman-Tytell medium without serum during the assay. CF was moderately sensitive to treatment with TLCK and TAME; however, its activity in serum and apparent molecular weight distinguish it from a moiety obtained from BCG-activated murine macrophages and previously described. A rabbit heteroantiserum raised against highly purified necrosin, a product of the murine macrophage cell line J774.1, was extremely effective in neutralizing the biological activity of CF.