In guinea pigs, inhalation of cotton dust results in an acute pulmonary response with symptoms of increased breathing rate, cough, bronchoconstriction, and periods of apnea. These symptoms resemble those noted in individuals upon exposure to cotton and other organic dusts. A major contaminant of cotton dust is bacterial endotoxin. Because endotoxin, or lipopolysaccharide, is recognized to be a potent stimulator of tumor necrosis factor (TNF), it was postulated that TNF might be released in the lung following cotton dust exposure and associated with the pulmonary inflammatory response. Groups of guinea pigs were exposed to an atmosphere of 33 mg/m3 cotton dust for up to 6 h. At 3, 6, 7.5, and 24 h, lungs were isolated and lavaged to assess cell populations and production of TNF. Neutrophil infiltration was apparent by 3 h as was a marked increase in TNF in bronchial alveolar lavage fluid. Alveolar macrophages (AM) isolated at 3 h showed enhanced release of TNF upon in vitro culture when compared with those isolated at the other time points. AM were found to be primed to release TNF upon ex vivo stimulation with lipopolysaccharide. The greatest effect was noted with AM isolated 1.5 h after the 6-h cotton dust exposure. These results demonstrate the ability of cotton dust to cause release of TNF in the lung and suggest a role for TNF in the inflammatory response to cotton dust.