Three subpopulations of macrophages present in the lung, viz. pulmonary alveolar macrophages (PAM), pulmonary tissue macrophages (PTM), and macrophages in the bronchus-associated lymphoid tissue (BALT), were compared using enzyme-cytochemical and immunocytochemical methods. BALT macrophages can be distinguished from PAM and PTM on the basis of their enzyme reaction products. With three monoclonal antibodies, ED1, ED2, and ED3, that recognize different antigen determinants of rat macrophages, the three subpopulations could be clearly distinguished. PAM are ED1-positive and PTM are ED2-positive. The macrophage subpopulation scattered throughout BALT is ED1-positive; macrophages situated at the peripheral site of BALT, near artery and bronchus, are ED2-positive. No ED3-positive macrophages were observed either in the lung or in BALT. These results are discussed with respect to the possible relationship between the subpopulations of pulmonary macrophages.