The nuclear import factor p10 was cloned from Saccharomyces cerevisiae and found to be essential. The protein p10 can bind directly to several peptide repeat-containing nucleoporins. It also binds to the guanosine triphosphatase (GTPase) Ran in its guanosine diphosphate (GDP)-bound form and to karyopherin beta. Assembly of the karyopherin heterodimer on immobilized nucleoporin yielded cooperative binding of p10 and Ran-GDP. Addition of GTP to this pentameric complex led to dissociation of karyopherin alpha, presumably via in situ formation of Ran-GTP from Ran-GDP. Thus, p10 appears to coordinate the Ran-dependent association and dissociation reactions underlying nuclear import.