Alveolar macrophages (AM) play an important role in lung biology. In this study, we demonstrated that tracheal insufflation of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP-liposome) selectively depleted AMs in rats. Insufflation of a single dose of Cl2MDP-liposomes (80 microliters containing 1.34 mumol of Cl2MDP) but not liposomes containing phosphate-buffered saline resulted in > 70% depletion of AMs starting within 1 day and lasting for > 5 days after insufflation. There was a slight but significant intraalveolar inflammatory response. Insufflation of Cl2MDP also resulted in depletion of AMs; however, it caused cytoplasmic edema of alveolar epithelial cells as well. Depletion of AMs by Cl2MDP-liposomes markedly reduced the endotoxin-induced neutrophil (polymorphonuclear lymphocyte) recruitment and the release of tumor necrosis factor into the alveolar space, suggesting that endotoxin-induced neutrophil recruitment and tumor necrosis factor release were dependent on AMs. This AM-depleted animal model will be useful for studying the in vivo functions of AMs and their role in various physiological and pathological conditions.