The monoclonal antibody (mAb) ED1 is being used widely as a marker for rat macrophages. The distribution of the recognized antigen in tissues and isolated cells strongly supports this use as a macrophage marker, since the majority of macrophages are recognized and only seldomly are other cell types stained by mAb ED1. In the present study we further characterized the recognized antigen by a detailed description of the localization of the antigen and by determining biochemical and functional properties. We show that the antigen is expressed on the membranes of cytoplasmic granules, like phagolysosomes, as well as on the cell surface. The amount of ED1 expression in a single cell can be correlated to phagocytic activity of the respective cell type, but the mAb ED1 is not able to block latex phagocytosis or bacterial killing. The mAb ED1 appears to recognize a heavily glycosylated protein of 90,000-110,000 MW, depending on the cell type used as antigen source. A possible relation with other known lysosomal glycoproteins with a similar molecular weight is discussed.