NF-kappaB transcription factors are retained in the cytoplasm in an inactive form until they are activated and rapidly imported into the nucleus. We identified importin alpha3 and importin alpha4 as the main importin alpha isoforms mediating TNF-alpha-stimulated NF-kappaB p50/p65 heterodimer translocation into the nucleus. Importin alpha3 and alpha4 are close relatives in the human importin alpha family. We show that importin alpha3 isoform also mediates nuclear import of NF-kappaB p50 homodimer in nonstimulated cells. Importin alpha3 is shown to directly bind to previously characterized nuclear localization signals (NLSs) of NF-kappaB p50 and p65 proteins. Importin alpha molecules are known to have armadillo repeats that constitute the N-terminal and C-terminal NLS binding sites. We demonstrate by site-directed mutagenesis that NF-kappaB p50 binds to the N-terminal and p65 to the C-terminal NLS binding site of importin alpha3. In vitro competition experiments and analysis of cellular NF-kappaB suggest that NF-kappaB binds to importin alpha only when it is free of IkappaBalpha. The present study demonstrates that the nuclear import of NF-kappaB is a highly regulated process mediated by a subset of importin alpha molecules.