U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100 -144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin ␣/ and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin ␣. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin ␣/ and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin  binding domain of importin ␣. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin ␣/ and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.Cellular activities in eukaryotic cells are coordinated via the continuous and bi-directional transport of macromolecules between the nucleus and the cytoplasm, which occurs through the nuclear pore complexes (NPCs).1 The translocation of proteins through the NPCs is mediated by an active and selective mechanism that is controlled by saturable receptors and signals that are termed nuclear localization signals (NLSs) and nuclear export signals (reviewed in Refs. 1 and 2).The best characterized active nuclear protein import is mediated by a basic type NLS, referred to as "classical NLS," and which contains one or more clusters of basic amino acids. The import of substrates containing classical NLS such as SV40 large T antigen is initiated by the formation of an NLS-dependent complex with importin ␣/ in the cytoplasm, which is referred to as the nuclear pore-targeting complex. Importin ␣ recognizes the NLS and binds to importin  via its N-terminal sequences, which are rich in basic amino acids, and is referred to as the importin  binding (IBB) domain (3). Importin  accounts for the targeting of the complex to the NPC. In addition to this classical import pathway, several different types of pathways have been identified, which involve importin  or importin  family members that bind directly to their cognate cargoes without importin ␣ (4 -6).It has been shown that there are at least five different forms of importin ␣ in the human and mouse that display significantly different tissue expression patterns (7, 8). It has been also shown that these importin ␣ isoforms interact differentially with specific NLS (9 -11). As evidenced by the primary sequences, these molecules can be grouped into three subfamilies, which are referred to Rch1, NPI-1, and Qip1, and which show ϳ50% amino...