To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-kappa B, we stimulated human vascular endothelial cells with tumor necrosis factor-alpha in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-kappa B activation by tumor necrosis factor-alpha. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-kappa B, suggesting tonic inhibition of NF-kappa B under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-kappa B inhibitor, I kappa B alpha, by preventing its degradation from NF-kappa B. NO also increased the mRNA expression of I kappa B alpha, but not NF-kappa B subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the I kappa B alpha promoter suggested transcriptional induction of I kappa B alpha by NO. We propose that the induction and stabilization of I kappa B alpha by NO are important mechanisms by which NO inhibits NF-kappa B and attenuate atherogenesis.