The human nuclear and cytoplasmatic protein Ki-1/57 is associated with protein kinase activity and gets phosphorylated on serine and threonine residues upon cellular activation. Ki-1/57 shares 41% of sequence identity and 67% of sequence similarity with the human protein CGI-55, suggesting that both proteins could be paralogs. CGI-55 has been described as a RNA binding protein that binds to 3’-region of the RNA encoding the type-1 plasminogen activator inhibitor (PAI-1). In recent studies, we explored the yeast two-hybrid system and found that both Ki-1/57 and CGI-55 interact with the several common proteins involved in transcriptional regulation, including CHD3, Topors, Daxx and p53. Furthermore, several of the identified Ki-1/57 interacting proteins are involved in RNA metabolism (CIRBP, SFRS9, SF2/p32). Although neither Ki-1/57 nor CGI-55 posses any classical RNA binding domains, they both share two conserved RGG box motifs, which have been predicted as motifs involved in RNA binding. In order to investigate the RNA binding properties of Ki-1/57 we performed Eletrophoretic Mobility Shift Assays (EMSA) using the RNA homo polymers poly-U, poly-A, poly-C, poly-G and an AU-rich oligonucleotide as ³²P-radiolabeled probes. We found that the recombinant fusion protein GST-Ki-1/57 binds selectively to the poly-U probe. When we used several truncated protein constructions of Ki-1/57, we observed that the larger fusion construction 6xHis-Ki-1/57(122-413), which contains two RGG box motifs, binds with higher affinity to the U-rich probe than two smaller constructs, which contain only one RGG-box motif each. Furthermore, when we pre-incubated the GST-Ki-1/57 protein, coupled to glutathione beads, with nuclear or cytoplasmic fractions of human L540 cells, we observed different interaction profiles for the five RNA probes in the EMSA. This could suggest that Ki-1/57 may bind to other proteins or RNA binding protein complexes in vivo. In summary, our findings point to a possible role in RNA metabolism for the regulatory protein Ki-1/57.