Nucleolin is a major nucleolar protein in mammalian cells that is thought to be involved in ribosome biogenesis. The discovery that nucleolin shuttles between the cytoplasm and the nucleus raises the possibility that it is also involved in transporting ribosomal or nuclear proteins to the nucleus. The three structural domains of nucleolin bear a striking resemblance to the domains of a previously identified yeast protein NSR1, although the two proteins do not share a high degree of sequence similarity. NSR1 specifically recognizes the nuclear localization sequence (NLS) of both the simian virus large T antigen (SV40 T-antigen) and the yeast histone H2B by ligand blot analysis, and is a candidate for a receptor involved in the initial stages of nuclear transport. We report here that nucleolin, either purified from Chinese hamster ovary (CHO) cells or expressed in yeast, also specifically recognizes the wild-type, but not a mutant, histone H2B nuclear localization sequence by ligand blot analysis. The NLS recognition site is located within the N-terminal domain of both proteins. In showing that nucleolin, a protein that moves between the cytoplasm and the nucleus, also has the ability to interact with nuclear localization signals, our data support the idea that shuttling nucleolar proteins play a role in nuclear transport.