Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.