We found by computer analysis that a putative yeast proteasome subunit gene named PRS3 that encodes a protein very similar to subunit C5 of rat and human proteasomes is located immediately 3' to the ERD2 gene of Saccharomyces cerevisiae. The similarity of the primary structures of the two suggests that this subunit may have a common function in proteasomes of all eukaryotes. The protein, deduced from the open reading frame of PRS3, consists of 242 amino acid residues with a calculated molecular weight of 27,077. Chromosomal disruption of the PRS3 gene created a recessive lethal mutation. Physical mapping by hybridization to intact S. cerevisiae chromosomal DNA showed that the PRS3 gene is located on chromosome II, unlike two other subunit genes, PRS1 and PRS2, which are located on chromosomes XV and VII, respectively. These findings indicate that the PRS3 protein is a subunit of yeast proteasomes that is essential for cell viability.