URP2 was cloned as a multicopy suppressor of several temperature-sensitive mutations defective in RNA polymerase III-dependent transcription, but without effect on mutations affecting RNA polymerase I or II. This single-copy gene encodes a hydrophilic polypeptide of 121 amino acid residues with a predicted molecular mass of 13.9 kDa and a basic isoelectric point of 9.7. URP2 is a highly expressed gene, judging from its abundant messenger RNA and strong codon bias. The Urp2p protein is essential for cell growth, as shown by the lethal phenotype of the urp2::HIS3 null allele. Given its striking similarity to the S20 ribosomal polypeptide of rat (55% identical residues), Urp2p is in all likelihood the yeast form of this polypeptide. Both proteins are significantly related to S10, a component of the small ribosomal subunit of Escherichia coli that is known to operate as a transcriptional elongation factor. The latter observation suggests that the suppressor effect of URP2 may be due to a direct involvement of Urp2p in RNA polymerase III-dependent transcription. Alternatively, the overexpression of Urp2p could bypass a partial preribosomal RNA processing defect associated with RNA polymerase III mutants. URP2 was assigned to the left arm of chromosome VIII, and maps between DUR3 and YLF1. The latter gene product has homology to the E. coli gtp1 gene product, and may define a new family of putative GTP-binding proteins.