Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins α and β. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin α/β-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin α with importin β from cell extracts was strongly associated with import efficiency. These results indicate that the importin α/β-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins α and β.