Macrophage specific antigens were detected with fluorescein and 125I-labeled rabbit antibodies to anti-guinea pig macrophages on surface membranes of 85-100% of peritoneal exudate macrophages (PEM), 35-90% of alveolar macrophages (AVM), 35-50% of splenic macrophages, and 10% of peripheral blood monocytes. None was detected in bone marrow cells, the source of precursor macrophages. Phagocytosis in vitro was not accompanied by an increase of labeled cells in bone marrow, blood, or spleen. Neither trypsin nor neuraminidase reduced macrophage-specific antigens on surface membranes of PEM nor uncovered such antigens in greater quantity in mononuclear phagocytes of bone marrow, blood or spleen. Neuraminidase augmented the binding of 125I-labeled antimacrophage globulins (AMG) and normal rabbit globulins to monocytes/macrophages.