The transcription factor NF-kappaB regulates the expression of genes involved in cancer cell invasion, metastasis, angiogenesis, and resistance to chemotherapy. In normal cells NF-kappaB is maintained in the cytoplasm by protein-protein interaction with inhibitor IkappaBs. In contrast, in cancer cells a substantial amount of NF-kappaB is in the nucleus and constitutively activates target genes. To understand the mechanisms of constitutive NF-kappaB activation, we have analyzed the function of IkappaBalpha and IkappaBbeta in breast cancer cells. In most cases, constitutive NF-kappaB DNA binding correlated with reduced levels of either IkappaBalpha or IkappaBbeta isoforms. Overexpression of IkappaBalpha but not IkappaBbeta1 resulted in reduced constitutive DNA binding of NF-kappaB in MDA-MB-231 cells. Unexpectedly, IkappaBbeta1 overexpression moderately increased 12-O-tetradecanoylphorbol-13-acetate- and interleukin-1-inducible NF-kappaB DNA binding. 12-O-Tetradecanoylphorbol-13-acetate- and interleukin-1-induced transactivation by NF-kappaB, however, was lower in IkappaBbeta1-overexpressing cells. Mutants of IkappaBbeta1 lacking the C-terminal casein kinase II phosphorylation sites, which form a stable complex with DNA bound NF-kappaB without inhibiting its transactivation in other cell types, repressed the transactivation by NF-kappaB in MDA-MB-231 cells. Consistent with the results of transient transfections, the expression of urokinase plasminogen activator, an NF-kappaB target gene, was reduced in IkappaBbeta1-overexpressing cells. These results suggest that depending on the cell type, IkappaBbeta1 represses the expression of NF-kappaB-regulated genes by inhibiting either DNA binding or transactivation function of NF-kappaB.