An Rpn9-disrupted yeast strain, Δrpn9, whose growth is temperature sensitive with defective assembly of the 26S proteasome complex, was studied. This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type. Nondenaturing gel electrophoresis followed by activity staining revealed that Δrpn9 yeast cells had a higher activity of 20S proteasome than wild type and that in both Δrpn9 and wild-type cells treated with hydrogen peroxide, 20S proteasome activity was increased with a concomitant decrease in 26S proteasome activity. Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells. Taken together, these results suggest that the 20S proteasome degrades oxidized proteins without ubiquitination of target proteins.