To investigate the role of endotoxin in grain dust-induced airway inflammation, we reduced the endotoxin activity from extracts of corn dust (CDE), using three distinct methods, and determined the effect of endotoxin activity on the in vitro and in vivo inflammatory response to CDE. Escherichia coli lipopolysaccharide solution (LPS) and CDE solution were separated into > 100-kDa and < 100-kDa fractions by ultracentrifugation. Endotoxin activity was predominantly present in the > 100-kDa fractions of the LPS and CDE solutions. Charged-membrane filtration of the > 100-kDa fractions of LPS and CDE resulted in the reduction of endotoxin activity by 99.9 and 80%, respectively. Treatment of the > 100-kDa fractions of LPS and CDE with polymyxin B-coated beads reduced the endotoxin activity by 96 and 89%, respectively. The untreated > 100-kDa fractions of LPS and CDE caused significantly greater (P < 0.01) release of tumor necrosis factor-alpha (TNF-alpha) from THP-1 cells in vitro compared with its respective < 100-kDa fraction or either of the treated (charged filter or polymyxin B) > 100-kDa fractions. Similarly, mice exposed to either of the untreated > 100-kDa fractions of LPS or CDE by inhalation developed significantly greater (P < 0.01) concentrations of lavage neutrophils and TNF-alpha in the lavage fluid compared with mice exposed to the respective < 100-kDa fraction or either of the treated > 100-kDa fractions. These results indicate that endotoxin is primarily responsible for the in vitro and in vivo inflammatory response to CDE.