K homology (KH) domains are scaffolds for the binding of RNAs by the heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and its yeast ortholog, Hek2p. KH domains are remarkably conserved between mammals and yeast. To assess the binding activity for yeast RNA of the two proteins, we used full-length K protein and Hek2p as baits in the yeast three-hybrid system. All the unique RNA sequences bound by Hek2p and all but two bound by K protein represented different fragments of only two transcripts, encoded by the 18S and 25S ribosomal RNA genes. Most of them were transcribed from the antisense strand. The RNA-binding activity of K protein was significantly higher than that of Hek2p. These results and those from our previously published reports demonstrate that the specificity of target RNA recognition by both the K protein and Hek2p depends on both RNA-specific sequences and the structure of the protein. Both mammalian K protein and its yeast ortholog may be involved in the regulation of gene expression.