Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin. Yeast contain a homologue of karyopherin alpha named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I. To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin alpha homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera. Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose. Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII. Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin beta. The yeast alpha and beta karyopherin subunits were expressed in Escherichia coli and were purified. When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells. We propose that all karyopherins function as alpha/beta heterodimers.