Ribosome biogenesis in Saccharomyces cerevisiae occurs primarily in a specialized nuclear compartment termed the nucleolus within which the rRNA genes are transcribed by RNA polymerase I into a large 35 S rRNA precursor. The ensuing association/dissociation and catalytic activity of numerous trans-acting protein factors, RNAs and ribosomal proteins ultimately leads to the maturation of the precursor rRNAs into 25, 5.8 and 18 S rRNAs and the formation of mature cytoplasmic 40 and 60 S ribosomal subunits. Although many components involved in ribosome biogenesis have been identified, our understanding of this essential cellular process remains limited. In the present study we demonstrate a crucial role for the previously uncharacterized nucleolar protein Nop53p (Ypl146p) in ribosome biogenesis. Specifically, Nop53p appears to be most important for biogenesis of the 60 S subunit. It physically interacts with rRNA processing factors, notably Cbf5p and Nop2p, and co-fractionates specifically with pre-60 S particles on sucrose gradients. Deletion or mutations within NOP53 cause significant growth defects and display significant 60 S subunit deficiencies, an imbalance in the 40 S:60 S ratio, as revealed by polysome profiling, and defects in progression beyond the 27 S stage of 25 S rRNA maturation during 60 S biogenesis.