We have identified and characterized a 9S protein complex from a Xenopus ovary cytosolic subfraction (fraction A) that constitutes this fraction's activity in recognizing a model nuclear import substrate and docking it at the nuclear pore complex. Because of its function, the complex is termed karyopherin. The 54- and 56-kDa subunits of the complex are termed alpha 1 and alpha 2, respectively, and the 97-kDa subunit is termed beta. In an alternative approach we have identified karyopherin beta from a rat liver cytosolic subfraction A by using immobilized rat nucleoporin Nup98 in a single, affinity-based enrichment step. We have molecularly cloned and sequenced rat karyopherin beta. Comparison with protein sequence data banks showed no significant similarity to other known proteins. Using nitrocellulose-immobilized rat liver nuclear envelope proteins and nuclear import substrate as a ligand, we found Xenopus fraction A-dependent binding to at least three bona fide nucleoporins (Nup214, Nup153, and Nup98) and to a candidate nucleoporin with an estimated molecular mass of 270 kDa. We propose that these nucleoporins function as docking proteins for karyopherin-mediated binding of substrate in a nuclear import/export pathway across the nuclear pore complex.