To define the role of macrophages in regulating the lung's response to Escherichia coli endotoxin (lipopolysaccharide [LPS]), depletion of macrophages was accomplished by administration of dichloromethylene diphosphonate (clodronate) delivered via intratracheal (IT) and/or intravenous (IV) routes. Clodronate reduced the number of macrophages in lung lavage 48 h after either IT or IV administration, but combined IT + IV clodronate achieved the most profound depletion (90%). Although IT clodronate alone had little effect on the evolution of lung inflammation, combined IT + IV clodronate treatment decreased neutrophilic alveolitis 4 h after exposure to aerosolized LPS by 80% compared with mice treated with empty liposomes. This decrease was associated with impaired activation of nuclear factor (NF)- κ B and lower concentrations of tumor necrosis factor (TNF)- α in lung lavage fluid. Combined IT + IV clodronate markedly reduced lung NF- κ B activation and the intensity of neutrophilic alveolitis after intraperitoneal (IP) LPS; however, IV clodronate alone had no effect on NF- κ B activation in either liver or lung tissue or the development of neutrophilic alveolitis. We conclude that generalized macrophage depletion reduces NF- κ B activation, generation of cytokines, and neutrophilic lung inflammation in response to gram negative bacterial endotoxin. These findings define the role of the macrophage as a critical component for initiation of the NF- κ B–dependent innate immune response.