Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin α/β. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPα recognized both NLSs, with y-IMPα exhibiting higher affinity, m-IMPβ greatly enhanced the binding of m-IMPα to the T-ag and N1N2 NLSs, but y-IMPβ did not significantly affect the affinity of y-IMPα for the T-ag NLS. In contrast, y-IMPβ enhanced y- IMPα binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPα binding by m-IMPβ. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.