A hybridoma clone which secretes a macrophage (MΦ)‐specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate‐induced mouse peritoneal MΦ 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 MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ‐like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG_2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ 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 MΦ and, at least in part, exposed on the cell surface.