Importin beta can shuttle between the nucleus and cytoplasm through the nuclear pore complex (NPC). This study deals with the issue of how the energy is utilized during the NPC passage of importin beta. In chilled or ATP-depleted cells, importin beta was transported into the nucleus, while the nuclear export of importin beta was inhibited. Further, it was found that the nuclear export inhibition of importin beta is not due to nuclear retention via binding to nucleoporins or nuclear importin alpha. These data show that the nuclear export of importin beta involves energy-requiring step(s) in living cells.