In eukaryotes, karyopherin beta superfamily proteins mediate nucleocytoplasmic transport of macromolecules. We investigated the evolutionary and transcriptional patterns of these proteins using bioinformatics approaches. No obvious homologs were found in prokaryotes, but an extensive set of beta-karyopherin proteins was found in yeast. Among 14 beta-karyopherins of Saccharomyces cerevisiae, eight corresponded to their human orthologs directly without diversification, two were lost, and the remaining four proteins exhibited gene duplications by different mechanisms. We also identified beta-karyopherin orthologs in Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Xenopus tropicalis, Gallus gallus, and Mus musculus. beta-Karyopherins were ubiquitously but nonuniformly expressed in distinct cells and tissues. In yeast and mice, the titer of some beta-karyopherin transcripts appeared to be regulated both during the cell cycle and during development. Further virtual analysis of promoter binding elements suggested that the transcription factors SP1, NRF-2, HEN-1, RREB-1, and nuclear factor Y regulate expression of most beta-karyopherin genes. These findings emphasize new mechanisms in functional diversification of beta-karyopherins and regulation of nucleocytoplasmic transport.