Recent studies have advanced our knowledge about the signal transduction cascade involved in the activation of nuclear factor (NF) kappaB, including the identification and characterization of IkappaB kinases (IKKs). Although exposure to hydrogen peroxide (H2O2) in vitro can activate NF-kappaB, this response is not universal and depends on the cell type and transformation state. In this study, we examined the effects of H2O2 on IKKs and activation of NF-kappaB in primary normal human bronchial epithelial (NHBE) cells. Our results demonstrate that treatment with H2O2 increased IKK activity, phosphorylation, and ubiquitination of IkappaBalpha in NHBE cells. However, there was no significant proteolytic degradation of IkappaBalpha, nuclear translocation of p65, or NF-kappaB DNA binding activity in cells treated with H2O2. Treatment with H2O2 also inhibited tumor necrosis factor (TNF)-alpha-induced IkappaBalpha breakdown, NF-kappaB DNA binding activity, and NF-kappaB-dependent transcription but had no effect on TNF-alpha-induced IkappaBalpha phosphorylation or ubiquitination. Furthermore, treatment with H2O2 alone or in combination with TNF-alpha increased the levels of other ubiquitinated proteins in NHBE cells, suggesting general inhibition of proteasomal activity by H2O2. Taken together, these results demonstrate that in airway epithelial cells treatment with H2O2 has opposing effects on IKK activity and proteasomal degradation of IkappaBalpha, and suggest that H2O2 may suppress TNF-alpha-induced NF-kappaB- dependent gene expression.