When human alveolar macrophages (AM) lavaged from healthy donors were incubated in medium with or without lipopolysaccharide (LPS) or muramyl dipeptide, they released a factor(s) responsible for tumor cell killing. The activity of the tumor cytolytic factor(s), called TCF, was determined by radioactive release assay. Human AM released variable amounts of TCF into the culture medium without any stimulation, but the release was stimulated significantly by LPS (0.1 micrograms/ml) or muramyl dipeptide (1 micrograms/ml). Maximal production of TCF by the AM was detected in the supernatant after treatment for 3 hr with LPS, and the extent of TCF release correlated with the density of AM. In cultures with LPS, the ability of activated AM to secrete TCF was maintained for 48 hr but was lost by 96 hr. After its loss, the ability to produce TCF could be restored by a second treatment with LPS. Full expression of lysis by TCF to lyse tumor cells required its interaction with tumor cells for at least 24 hr. TCF destroyed human allogeneic tumor cell lines but did not affect nonneoplastic cell lines. TCF activity was resistant to treatment with protease inhibitors, superoxide dismutase, or catalase and to heating at 70 degrees for 1 hr, but it was labile on heating at 100 degrees for 10 min. The tumoricidal activity in the supernatant of activated human AM indicates a potential effector mechanism by which AM kill neoplastic cells.