A subtype of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like (ABC) DLBCL, depends on constitutive nuclear factor-kappaB (NF-kappaB) signaling for survival. Small molecule inhibitors of IkappaB kinase beta (IKKbeta), a key regulator of the NF-kappaB pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKKbeta inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would increase the killing of an ABC DLBCL cell line in the presence of a small molecule IKKbeta inhibitor. Two independent shRNAs targeting IKKalpha synergized with the IKKbeta inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKKalpha shRNAs blocked the classical rather than the alternative NF-kappaB pathway in ABC DLBCL cells, as judged by inhibition of IkappaBalpha phosphorylation. IKKalpha shRNA toxicity was reversed by coexpression of wild-type but not kinase inactive forms of IKKalpha, suggesting that IKKalpha may directly phosphorylate IkappaBalpha under conditions of IKKbeta inhibition. In models of physiologic NF-kappaB pathway activation by CARD11 or tumor necrosis factor-alpha, compensatory IKKalpha activity was also observed with IKKbeta inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKKalpha and IKKbeta, possibly through CARD11 inhibition.