Tumor necrosis factor alpha (TNF-alpha), originally defined by its antitumoral activity, is now recognized as a polypeptide mediator of inflammatory and cellular immune response. Recent studies have demonstrated that TNF-(alpha exists in the fluid of otitis media with effusion and, therefore, suggested its possible role in the pathogenesis of mucus hypersecretion. In this study, the effects of TNF-alpha on mucous glycoprotein (MGP) secretion from cultured chinchilla middle ear epithelial cells were examined, and TNF-alpha was found to stimulate MGP secretion in a time- and concentration-dependent manner. The action of TNF-alpha on MGP secretion was significantly and dose-dependently inhibited by TNF-alpha monoclonal antibody; this finding is suggestive of its specificity on MGP secretion. The addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperidine (H-7) to the culture significantly blocked TNF-alpha-induced MGP secretion, while the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) did not. This suggests that TNF-alpha stimulates MGP secretion via a protein kinase C-dependent mechanism.