bIn contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis. Ribosome biogenesis is an essential and highly complex process in all living organisms. Ribosomes are composed of numerous precisely assembled proteins and rRNA molecules; thus, they present a paradigm for RNA-protein recognition/binding and ribonucleoprotein (RNP) folding. In prokaryotes, the highly ordered process of ribosome assembly is relatively well defined and has been shown to involve orchestrated changes in rRNA conformation and protein binding steps (1). In contrast, very little is known about the mechanism of eukaryotic ribosome assembly. Despite the general similarity of eukaryotic and prokaryotic ribosomes, they differ substantially in their size, structure, and subunit compositions (including the numbers and sequences of proteins and rRNAs). Another key difference is that the process of maturation and assembly of eukaryotic ribosomes is a highly compartmentalized process which starts in the nucleolus and finishes in the cytoplasm (2). Factors that are not ribosome components but are involved in the ribosome maturation and assembly process also may differ between prokaryotes and eukaryotes. Production of eukaryotic ribosomes (including export from the nucleolus into the cytoplasm) requires highly synchronized synthesis of four rRNAs, about 80 ribosomal proteins, 150 proteins that serve as assembly factors, and 70 small nucleolar RNAs (snoRNAs) that direct modifications to rRNAs (3, 4). Advances in affinity purification methods using epitope-tagged proteins and mass spectrometry have allowed molecular characterization of several distinct RNP complexes corresponding to nucleolar preribosomes at different stages of assembly (5-7). However, this information was primarily derived from stud...