Nuclear factor (NF)-kappaB is an important regulator of inflammatory gene expression. Transcriptional regulation of Nos2, the inducible nitric-oxide synthase (iNOS) gene, is complex and not fully understood, but appears to be regulated in part by NF-kappaB. To further understand the role of NF-kappaB in Nos2 expression, we compared three functionally distinct NF-kappaB inhibitors on NF-kappaB transactivation and iNOS induction by rat C6 glial cells. Cytokine-induced activation of a consensus NF-kappaB-reporter gene was concentration-dependently inhibited by BAY 11-7082, MG-132, and helenalin. The rank order of potency was MG-132>helenalin>BAY 11-7082, with low concentrations of helenalin stimulating reporter gene activity. Cytokine-stimulated iNOS expression, measured by nitrite accumulation and in vitro l-citrulline production, was similarly reduced by exposing C6 cells to the NF-kappaB inhibitors. Surprisingly, activation of Nos2-reporter gene constructs containing the proximal 188 bp (containing one kappaB site) or proximal 94 bp (no kappaB site) of the rat promoter also was inhibited with the same rank order of potency. Interestingly, low concentrations of helenalin increased activity of both promoter constructs, while BAY 11-7082 poorly inhibited the 94-bp activity. This is the first report describing BAY 11-7082 and helenalin effects on iNOS expression in astroglia. Given the reported mechanism of actions for these inhibitors, cytokine-induced glial iNOS expression appears more sensitive to disruption of proteasome degradation and p65 function than modulation of IkappaB phosphorylation. These findings may foster the design of therapeutic agents aimed at NF-kappaB-associated pathways involved in neuroinflammation, especially iNOS expression.