1. In this study, we utilised a number of adenoviral constructs in order to examine the role of intermediates of the NF-κB pathway in the regulation of inducible nitric oxide synthase (iNOS) induction in rat aortic smooth muscle cells (RASMCs). 2. Lipopolysaccharide (LPS) stimulated a significant increase in iNOS induction and NF-κB DNA binding. These parameters were substantially reduced by overexpression of a wild-type Iκ-Bα adenoviral construct (Ad.Iκ-Bα), confirming a role for NF-κB in iNOS induction. 3. Infection with a dominant-negative IKKα adenoviral construct (Ad.IKKα(+/−)) did not significantly affect iNOS induction, NF-κB DNA binding or Iκ-Bα loss. Infection of RASMCs with adenovirus encoding a dominant-negative IKKβ (Ad.IKKβ(+/−)) essentially abolished iNOS induction and activation of the NF-κB pathway. 4. Pretreatment of RASMCs with a novel specific inhibitor of IKKβ, SC-514, significantly reduced iNOS induction, NF-κB DNA binding and I-κBα loss in a concentration-dependent manner. 5. In both RASMCs and human umbilical vein endothelial cells (HUVECs), infection with Ad.IKKβ(+/−) also inhibited COX-2 expression in response to LPS. However, Ad.IKKα(+/−) was again without effect. 6. These data suggest that IKKβ plays a predominant, selective role in the regulation of NF-κB-dependent induction of iNOS in RASMCs.