We report here the identification and characterization of a novel nucleolar RNA binding protein, referred to as Nop25 as based on its predicted molecular size and subcellular location. Nop25 homologues were widely discovered in diverse vertebrate species as hypothetical proteins, but not found in yeasts, plants and prokaryotic organisms. Nop25 was ubiquitously expressed in adult mouse organs and constitutively during mouse embryogenesis. Indirect immunofluorescence analysis with an anti-Nop25 antibody, as well as an experiment using a GFP-fused protein, demonstrated that Nop25 was localized in the nucleolus. Treatment of the cells with a low doses of actinomycin D caused Nop25 to translocate to the periphery of the nucleolus, suggesting that nucleolar localization of Nop25 is associated with rRNA transcription. Treatment of COS7 cells with RNase A resulted in a complete dissociation of Nop25 from the nucleolus, while in vitro binding assay demonstrated that Nop25 could bind directly to single-stranded nucleic acids. Further characterization of associated RNA molecules with Nop25 using immunoprecipitation experiment showed that Nop25 might bind to 28S rRNA. Studies on this novel nucleolar RNA binding protein may provide new information on the intricate nucleolar machinery as related to the transcription and processing of rRNA molecules and/or the subsequent assembly and maturation of ribosomes.