We have identified a yeast gene encoding a protein structurally similar to mammalian nucleolin. The gene was previously cloned as a cold shock-inducible gene and found to be identical to yeast NSR1 gene, which encodes a protein that has been reported to bind sequences required for nuclear localization of protein. The carboxyl-terminal half of NSR1, consisting of two tandemly repeated putative RNA-binding domains and a glycine/arginine-rich domain, has 37% amino acid sequence identity with the same part of mammalian nucleolin, while no sequence similarities are found between their amino-terminal regions. Although a null mutation of the NSR1 gene was not lethal, it caused a severe defect on growth. Pulse-labeling analysis revealed that the nsr1 strain had reduced levels of 18 S rRNA and accumulated 35 S pre-rRNA compared with the wild-type strain. The level of 25 S rRNA was also slightly reduced in the nsr1 strain. Pulse-chase labeling experiments showed slow processing of 35 S pre-rRNA and impaired methylation of 18 S rRNA. The ratio of 40 S to 60 S ribosomal subunits in the nsr1 strain is significantly reduced and is consistent with impaired synthesis of 18 S rRNA. The results indicate that NSR1 is involved in pre-rRNA processing and ribosome biosynthesis in yeast.