In eukaryotes, pre‐rRNA processing depends on a large number of nonribosomal trans‐acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two‐hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source‐conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose‐containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre‐rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two‐hybrid system, but its depletion affects the exosome function. In pull‐down assays, protein A‐tagged Nop53p coprecipitated the 27S and 7S pre‐rRNAs, and His–Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre‐rRNA processing.