A stable human macrophage hybridoma was established by somatic cell fusion between human peripheral blood monocyte-derived macrophages and an 8-azaguanine resistant clone of a human histiocytic lymphoma cell line U-937 (clone U-937-F9). The hybrid cell line (F9P) exhibited typical macrophage-like morphology and had 30 more chromosomes than U-937-F9 cells. Its macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mo-2 and LEU-M3 antigens on the cell surface, and the demonstration of phagocytic activity. Furthermore, when stimulated with lipopolysaccharide (LPS), this cell line could secrete a considerable amount of a cytotoxic factor (CTF). Distinct from the hybrid cell line, the parental U-937-F9 cells expressed neither Mo-2 nor LEU-M3 antigens on the cell surface, did not show phagocytic activity, and their culture supernatants did not show cytotoxic activity even after LPS stimulation. The activity of CTF in the culture supernatant of the LPS-stimulated hybrid cells could not be neutralized with anti-tumor necrosis factor, anti-interleukin-1, or anti-lymphotoxin antibodies. The CTF had a relative molecular mass of 45-60 x 10(3) daltons as determined by gel filtration on a column of Superose 12, and an isoelectric point of 5.1. The cytotoxic activity was also induced when the hybrid cells were stimulated with the concentrated supernatants of a human T-cell hybridoma containing macrophage activating factor for cytotoxicity or with LP3 tumor cells which were used as target cells.