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Airway intra-luminal macrophages (AI-LM) are a little-studied subpopulation of pulmonary macrophages that are located on the surfaces of the conducting airways of the lower respiratory tract. In this study, we: (1) developed a flow cytometric approach by which AI-LM can be viably isolated in high purity from cell suspensions obtained by airway washings; (2) comparatively examined various functional, biochemical, biophysical, and morphologic features of the rat's AI-LM and alveolar macrophage (AM) phenotypes, and (3) investigated the origin of the AI-LM in the rat. Airway cells were harvested from the tracheas of adult Fischer-344 rats, and AM were obtained from the lungs by conventional bronchopulmonary lavage or via prosthetic airway circuits that supplanted the removed tracheas. Flow cytometric analyses of lavaged airway cells revealed that the AI-LM fell within the range of the electro-optical phenotype of AM, and subsequent cell-sorting experiments demonstrated that virtually all viable AI-LM could be sorted from contaminating airway epithelial cells in > 95% purity based on their electro-optical characteristics, e.g., electronic volume, axial light loss, 90° light scatter, and blue and green autofluorescence signals. In Fcγ receptor-mediated phagocytic studies, ˜90% of AM engulfed opsonized erythrocytes (EIgG) whereas only 60% of the AI-LM were able to do so. Comparisons of the numbers of EIgG in phagocytic AM and in phagocytic AI-LM indicated the AI-LM were less phagocytic. Densitometrie analyses of sorted AI-LM and of sorted AM stained for acid phosphatase, nonspecific esterase, and β-glucuronidase indicated that the activities of these enzymes were generally less in the AI-LM than in the AM. Morphometric comparisons of sorted AM and of sorted AI-LM showed that the AI-LM were generally larger than the AM and that the surfaces and nuclei of the AI-LM were more regular than those of the AM. The AI-LM were found to strongly label with the monoclonal antibody EDI, which recognizes an antigen on the surfaces of rat AM, but the AI-LM did not label with the monoclonal antibody ED2, which recognizes an antigen on the surfaces of rat peribronchial and pulmonary perivascular macrophages. Over the course of alveolar phase clearance of a lung burden of polystyrene microspheres, the frequency distributions of the particles in AI-LM and in AM were found to be virtually identical. The results from these studies indicate that essentially all AI-LM are translocating AM, and they suggest that a substantial fraction of the AM undergo a transition to a functionally less active or effete state over the course of their removal from the lung as AI-LM.