Nuclear import of classical nuclear localization sequence-bearing proteins is mediated by karyopherin α/β1 heterodimers. A second nuclear import pathway, mediated by karyopherin β2 (transportin), recently was described for mRNA-binding proteins. Here we report the cloning and characterization of human karyopherin β3, which may be involved in a third pathway for nuclear import. Karyopherin β3 was localized mainly to the cytosol and the nucleus, particularly the nuclear rim. It bound to several of the repeat-containing nucleoporins (Nup358, Nup214, Nup153, Nup98, and p62) in overlay and solution-binding assays and was competed away by karyopherin β1. For Nup98, we localized this binding to the peptide repeat-containing region. Karyopherin β3 contains two putative Ran-binding homology regions and bound to Ran-GTP in a solution-binding assay with much higher affinity than to Ran-GDP. Furthermore, it interacted with two ribosomal proteins in an overlay assay. We suggest that karyopherin β3 is a nuclear transport factor that may mediate the import of some ribosomal proteins into the nucleus.