Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-kappaB) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-kappaB activation and consequent iNOS gene expression, we studied the effects of NO donors [(+/-)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (NOR3) and sodium nitroprusside] on interleukin (IL)-1beta-induced NF-kappaB activation and IkappaB-alpha degradation and subsequent iNOS expression in rat VSMCs. Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1beta-induced iNOS mRNA and protein expression. Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappaB site of rat iNOS promoter as a probe showed that NOR3 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation, as demonstrated with immunocytochemical study. These effects were independent of guanylate cyclase activation; an inhibitor of soluble guanylate cyclase (1H-oxadiazolo-1,2,4-[4,3-alpha]quinoxaline-1-one) had no effect on NOR3-induced inhibition of NF-kappaB activation or iNOS mRNA expression by IL-1beta, and a cGMP derivative (8-bromo-cGMP) failed to mimic the effects of NO donors. Western blot analysis using anti-IkappaB-alpha and anti-phospho-IkappaB-alpha antibodies revealed that IL-1beta induced a transient degradation of IkappaB-alpha preceded by a rapid appearance of phosphorylated IkappaB-alpha, both of which were completely blocked by NOR3. A proteasome inhibitor (MG115) blocked IL-1beta-induced transient degradation of IkappaB-alpha and stabilized the appearance of phosphorylated IkappaB-alpha stimulated by IL-1beta. NOR3 inhibited the appearance of IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of MG115. Our results indicate that an inhibitory action by NO on cytokine-induced NF-kappaB activation and iNOS gene expression is due to its direct blockade on phosphorylation and subsequent degradation of IkappaB-alpha via the cGMP-independent pathway in rat VSMCs.