Previous studies demonstrated that theophylline modulates NF-kappaB activation in mast cells and pulmonary epithelial cells. We examined whether or not this modulation of NF-kappaB activation by theophylline is due to inhibition of the degradation of the IKBalpha protein, which suppresses NF-kappaB activation. TNF-alpha-induced NF-kappaB activation in a human pulmonary epithelial cell line (A549) was evaluated by Western blotting and a chloramphenicol acetyltransferase (CAT) assay. Expression of the IkappaBalpha protein was evaluated by Western blotting. Western blotting of nuclear extracts of A549 cells demonstrated that theophylline suppresses NF-kappaB-p65 nuclear translocation. The CAT assay indicated that NF-kappaB-dependent reporter gene expression is inhibited in A549 cells pretreated with theophylline. Western blotting of cytoplasmic extracts of A549 cells revealed that this inhibition was linked to theophylline-induced protection of expression of the IkappaBalpha protein. Moreover, theophylline inhibited interleukin-6 production induced by TNF-alpha in A549 cells. These findings are consistent with the idea that theophylline suppresses the production of proinflammatory cytokines via inhibition of NF-kappaB activation through protection of the IkappaBalpha protein.