F4/80, a mouse macrophage-specific membrane marker defined by a rat monoclonal antibody, was precipitated by a rabbit antiserum raised against partially purified mouse antigen. The antiserum, when tested against a variety of mouse tissues and cells, bound only to, and was cytotoxic for, macrophages, and it precipitated a similar macrophage-specific protein from rat cells. The F4/80 antigen is a glycoprotein of apparent molecular weight (MW) 150,000, and was labelled biosynthetically with [14C]glucosamine. Neuraminidase treatment removed small amounts of sialic acid, and tunicamycin and 2-deoxyglucose both inhibited antigen synthesis. Pulse/chase labelling with [35S]methionine demonstrated a precursor of 110,000 MW. Proteinase treatment of intact cells cleaved the molecule to an initial 100,000, and then to an 80,000 MW fragment. Without reduction, the MW of the molecule was unchanged by proteinases. These studies indicate that the F4/80 antigen consists of at least two domains linked by disulphide bridges, of MW 80,000 and 20,000. Both domains are extracellular.