We investigated the role of neutrophils in the development of endotoxin-induced airway disease via systemic neutrophil depletion of C3H/HeBFeJ mice and coincident inhalation challenge with lipopolysaccharide (LPS) over a 4-wk period. Mice were made neutropenic with intraperitoneal injections of neutrophil antiserum before and throughout the exposure period. Experimental conditions included LPS-exposed, antiserum-treated; LPS-exposed, control serum-treated; air-exposed, antiserum-treated; and air-exposed, control serum-treated groups. Physiological, biological, and morphological assessments were performed after a 4-wk exposure and again after a 4-wk recovery period. After the 4-wk exposure, LPS-induced inflammation of the lower airways was significantly attenuated in the neutropenic mice, although airway responsiveness (AR) to methacholine (MCh) remained unchanged. After the recovery period, LPS-exposed neutrophil-replete mice had increased AR to MCh when compared with the LPS-exposed neutropenic animals. Morphometric data indicate that the 4-wk exposure to LPS leads to a substantial expansion of the subepithelial area of the medium-sized airways (90–129 μm diameter) in nonneutropenic mice but not neutropenic mice, and this difference persisted even after the recovery period. Expression of bronchial epithelial and subepithelial transforming growth factor-β1 (TGF-β1) was diminished in the challenged neutropenic mice compared with the neutrophil-sufficient mice. These studies demonstrate that neutrophils play a critical role in the development of chronic LPS-induced airway disease.