The IkappaB family of proteins regulates NF-kappaB-dependent transcription by inhibiting DNA binding and localizing these factors to the cell cytoplasm. IkappaBalpha does this by shifting the balance between nuclear import of Rel proteins and their export from the nucleus. Here we show that, unlike IkappaBalpha, IkappaBbeta and IkappaBepsilon appear to sequester p65 or c-Rel in the cytoplasm by inhibiting nuclear import. Furthermore, because IkappaBbeta does not undergo nucleocytoplasmic shuttling, it cannot remove nuclear proteins like IkappaBalpha does. We conclude that the mechanism of action differs among IkappaB family members.