Stable activated macrophage hybridomas were generated by somatic cell fusion between Propionibacterium acnes-induced peritoneal exudate cells and NS-1 myeloma cells. Five cell lines were obtained and each was cloned by limiting dilution; 59 clones were obtained. The cells of 2 clones (MP4-4 and MP4-8) which adhered to the culture dishes were selected for further analysis. These hybridomas exhibited non-specific esterase and beta-galactosidase intracellularly, and asialo GM1, Mac-1, Ia antigens and Fc-receptors on their cell surface. They did not, however, show phagocytic activity or secrete lysozyme. These hybridomas (MP4-4 and MP4-8) secreted the cytotoxic factor without any stimulation. Furthermore strong cytotoxic activity was found in ascites and sera from nude mice inoculated with these hybridomas. These activated macrophage hybridomas should be very useful in studies on cancer immunology and the physiology of macrophages.