Using truncated forms of recombinant yeast karyopherins alpha and beta in in vitro binding assays, we mapped the regions of karyopherin alpha that bind to karyopherin beta and the regions of karyopherin beta that interact with karyopherin alpha and with Ran-GTP. Karyopherin alpha's binding region for karyopherin beta was localized to its N-terminal domain, which contains several clusters of basic residues, whereas karyopherin beta's binding region for karyopherin alpha was localized to an internal region containing two clusters of acidic residues. Karyopherin beta's binding region for Ran-GTP overlaps with that for karyopherin alpha and comprises at least one of the two acidic clusters required for karyopherin alpha binding in addition to further downstream determinants not required for karyopherin alpha binding. Overexpression in yeast of fragments containing either karyopherin beta's binding region for alpha and Ran-GTP or karyopherin alpha's binding region for beta resulted in sequestration of most of the cytosolic karyopherin alpha or karyopherin beta, respectively, in complexes containing the truncated proteins. As these binding region-containing fragments lack other domains required for function of the corresponding protein, the overexpression of either fragment also inhibited in vivo nuclear import of a model reporter protein as well as cell growth.