Nuclear import of conventional nuclear localization sequence (NLS)-containing proteins initially involves recognition by the importin (IMP)␣The entry of karyophilic proteins into the nucleus through the nuclear pore complex (NPC) 1 is effected by specific targeting signals called nuclear localization sequences (NLSs) (1, 2), and is a receptor-mediated (3, 4), energy-dependent (5, 6) process. The key factors involved are members of the NLS-recognizing importin/karyopherin family (7-11), the monomeric GTPase Ran/TC4 (12, 13), and auxiliary proteins such as NTF2/p10 (14, 15). In the first step, the NLS-containing protein is recognized by the importin (IMP) heterodimer through the NLS-binding IMP␣ subunit (3, 7, 9) and targeted to the NPC through the affinity of the IMP subunit (8, 10, 11, 16) for NPC components (17, 18). In the second step requiring cytoplasmic RanGDP (19,20), the transport complex is translocated through the NPC (21), and IMP␣ and the NLS-bearing protein are released into the nucleoplasm through the action of Ran GTP (19). Alternative signal-mediated nuclear import pathways have recently been identified, where either IMP itself (22-24) or related homologs (25-27) fulfill the role of both IMP␣ and - in binding NLSs and targeting them to the NPC (25,26,28).Although NLS receptors from different species share structural and functional homology, experimental evidence suggests that nuclear import in plant cells has unique features compared with that in other eukaryotes. In contrast to the latter, in vitro transport in plant cells appears not to be inhibited by the nucleoporin-binding lectin wheat germ agglutinin and to occur at low temperature and in the absence of exogenously added cytosol (see Refs. 29 and 30). In addition, the NLS-binding IMP␣ subunit from Arabidopsis thaliana (At-IMP␣) shows nuclear envelope association (30,31) in similar fashion to IMP in mammalian and other cell systems (8, 10, 11); IMP␣ in mammalian and yeast systems shows predominantly nucleoplasmic location as well as cell cycle-dependent localization in either cytoplasm or nucleus in Drosophila (32). A linkage of At-IMP␣ with the cytoskeleton has also recently been demonstrated, with a mechanistic role in nuclear import surmised (33). Because of these novel properties, we set out to quantitate the NLS binding properties of At-IMP␣ for the first time using an ELISA-based assay (34, 35). We find that At-IMP␣ binds NLSs of different types with high affinity independent of an IMP subunit, in contrast to the IMP␣ subunits from mouse and yeast, which require their respective IMP subunits to achieve high affinity binding (21, 34 -36). At-IMP␣, together with Ran/ TC4 and NTF2 and in the absence of IMP, was able to mediate nuclear import in vitro to levels comparable with those mediated by mouse IMP␣/ (m-IMP␣/). m-IMP␣ was unable to mediate nuclear import in the absence of m-IMP. At-IMP␣ thus shows unique properties, being able to fulfill both NLS recognition and nuclear import in the absence of IMP.