IkappaB kinase-1 and IkappaB kinase-2 (IKK1 and IKK2; also called IKKalpha and IKKbeta, respectively) are part of the signal complex that regulates NF-kappaB activity in many cell types, including fibroblast-like synoviocytes (FLS). We determined which of these two kinases is responsible for cytokine-induced NF-kappaB activation in synoviocytes and assessed the functional consequences of IKK1 or IKK2 overexpression and inhibition. FLS were infected with adenovirus constructs encoding either wild-type (wt) IKK1 or IKK2, the dominant negative (dn) mutant of both kinases, or a control construct encoding green fluorescence protein. Analysis of the NF-kappaB pathway revealed that cytokine-induced IKK activation, IkappaB degradation, and NF-kappaB activation was prevented in cells expressing the IKK2 dn mutant, whereas baseline NF-kappaB activity was increased by IKK2 wt. In addition, synthesis of IL-6 and IL-8, as well as expression of ICAM-1 and collagenase, was only increased by IKK2 wt, and their cytokine-induced production was abrogated by IKK2 dn mutant. However, the IKK1 dn mutant did not inhibit cytokine-mediated activation of NF-kappaB or any of the functional assays. These data indicate that IKK2 is the key convergence pathway for cytokine-induced NF-kappaB activation. Furthermore, IKK2 regulates adhesion molecule, matrix metalloproteinase, and cytokine production in FLS.