A hybridoma clone which secretes a macrophage (M phi)-specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate-induced mouse peritoneal M phi with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence-activated cell sorter analysis. F4/80 binds to mouse M phi from the peritoneal cavity or other sources, blood monocytes, M phi derived from bone marrow precursors in culture and M phi-like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to M phi via Fc receptors, is not cytotoxic and is of the rat IgG2b subclass. Since F4/80 binds to all M phi defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the M phi in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the M phi and, at least in part, exposed on the cell surface.