Nucleolin (NCL) is one of the most abundant nucleolar proteins of exponentially growing eukaryotic cells. It is known to interact only transiently with rRNA and preribosomal particles and not to be detectable in mature cytoplasmic ribosomes, and is believed to function as multi-protein complexes during ribosome biogenesis and maturation. However, those multiprotein complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. Here we report isolation of NCL-binding protein complex and its proteomic characterization with the use of an analytical method based on matrix-assisted laser desorption/ionization-time of flight analysis coupled with searching peptide mass databases. The NCL-binding protein complex was isolated by immunoprecipitation with anti-Flag antibody from human kidney 293 cells that were transfected with the Flag-tagged NCL gene, and showed RNA integrity for holding their protein constituents. Interaction between NCL and its binding complex was disrupted by an RNA oligonucleotide with a NCL recognition element, indicating that NCL binds to the ribonucleoprotein (RNP) complex mainly through the sequence specific protein-RNA interaction. We confirmed that an RNA-binding domain of NCL alone was sufficient to hold the entire NCL-binding RNP complex, indicating the strict binding specificity of NCL to the isolated RNP complex in 293 cells. We identified forty ribosomal proteins from both the large and small subunits, and twenty nonribosomal proteins. These results together suggest that the isolated NCL-binding RNP complex is a preribosomal particle present in the nucleolus of 293 cells.