The transcription factor nuclear factor-B (NF-B) has been implicated in inflammatory and proliferative vascular mechanisms. Activated NF-B has been documented in human atherosclerotic lesions, and its activation in human vascular smooth muscle cells (SMC) by cytokines has been reported. However, intracellular mechanisms mediating NF-B activation in human SMC are poorly understood. The aim of this study was to explore the potential role of reactive oxygen species and oxidant stress as signaling events in cytokine-induced NF-B activation. Western blot analysis revealed the presence of inhibitory protein I-B in resting human aortic SMC, which was rapidly phosphorylated and degraded on exposure to interleukin-1 (IL-1) followed by NF-B translocation to the nucleus. IL-1 had no effect on two measures of intracellular oxidant stress, fluorescence generated by the oxidation of 2',7'-dichlorodihydrofluorescin to dichlorofluorescein (DCF) or changes in intracellular sulfhydryl content. N-acetylcysteine (NAC) a membrane-permeant antioxidant, which augmented intracellular sulfhydryl content and inhibited H₂O₂-induced DCF fluorescence, had no effect on cytokine-induced NF-B activation. In contrast to NAC, the metal chelators pyrrolidine dithiocarbamate and diethyldithiocarbamate attenuated IL-1-induced NF-B activation but had no effect on intracellular sulfhydryl content. Treatment of the cells with the oxidant H₂O₂ caused an increase in DCF fluorescence and decreased intracellular sulfhydryl content but had no effect on I-B or NF-B. In conclusion, this study suggests that oxidant stress may not play a major role in cytokine-induced activation of NF-B in human aortic SMC and that oxidants may not be primary activators of NF-B in these cells.