In eukaryotes, karyopherin  superfamily proteins mediate nucleocytoplasmic transport of macromolecules. We have investigated the evolutionary and transcriptional patterns of these proteins using bioinformatics approaches. No obvious homologs are found in prokaryotes, but an extensive set of beta-karyopherin proteins is found in yeast. Among 14 beta-karyopherins of S. cerevisiae, 8 correspond to their human orthologs directly without diversification, 2 are lost, and the remaining 4 proteins exhibit gene duplications by different mechanisms. We have also identified beta-karyopherinorthologs in C.elegans, D.melanogaster, D.rerio, X.tropicalis, G.gallus and M.musculus. Beta-karyopherins are ubiquitously but nonuniformly expressed in distinct cells and tissues. In yeast and mice, the titer of some beta-karyopherin transcripts appears to be regulated both during the cell cycle and during development. Further virtual analysis of promoter binding elements suggests that the transcription factors SP1, NRF-2, Hen-1, RREB-1 and NF-Y regulate most beta-karyopherin genes expression. These findings emphasize new mechanisms in functional diversification of beta-karyopherins and regulation of nucleocytoplasmic transport.