The redox state has been shown to regulate a variety of biochemical functions including cellular proliferation. Previous studies from our laboratory and others have shown that the binding of many transcription factors to their cognate DNA sequences is sensitive to the redox environment. Therefore, it is likely that redox status serves as an additional regulatory control for the activity of transcription factors and that this may mediate the redox regulation of proliferation. To assess this possibility, the influence of altering the redox state on NF-kappaB-regulated gene expression was studied. A more-reducing environment favored higher levels of expression of gro, an endogenous gene associated with proliferation, when the redox levels were changed either naturally by altering culture density or chemically by treatment with modulators of glutathione synthesis. Furthermore, nuclear runoff studies showed that a more-reducing redox increased transcription of gro. In order to ascertain the singular effect of the redox state on the activity of NF-kappaB, expression of a secreted alkaline phosphatase (SEAP) reporter gene solely under the control of an NF-kappaB response element was measured under varying redox conditions. Changes in the redox state modulated the expression of this reporter system. Taken together, these results suggest the involvement of a redox mechanism regulating signaling events operating through the control of gene expression by transcription factors.