A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995) EMBO J. 14, 5367-5378). The second one, encoded by YRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport.