We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expres-sion is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1β-induced iNOS gene expression, NF-KB activation, and NF-KB inhibitor (IKB)-α degradation in cultured rat VSMCs. N2733 dose-dependently (10-100μM) inhibited IL-1β-stimulated NO production, and decreased IL-1β-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1β-induced NF-KB activation and its nuclear translocation. Western blot analyses involving anti-IKB-α and anti-phospho IKB-α antibodies showed that IL-1β induced transient degradation of IKB-α preceded by the rapid appearance of phosphorylated IKB-α, both of which were markedly blocked by N2733. N2733 blocked IL-1β-induced phosphorylated IKB-α even in the presence of a proteasome inhibitor (MG115). hmnunoblot analysis involving anti-IKB kinase (IKK)-α and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1β-induced IKK-α phosphorylation, whereas N2733 had no inhibitory effect on IL-1β-stimulated p42/p44 MAP kinase or p3S MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1β-induced NF-KB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-α activation, and subsequent phosphorylation and degradation of IKB-α in rat VSMCs.