Rpn6p is a component of the lid of the 26S proteasome. We isolated and analyzed two temperature-sensitive rpn6 mutants in the yeast, Saccharomyces cerevisiae. Both mutants showed defects in protein degradation in vivo. However, the affinity-purified 26S proteasome of the rpn6 mutants grown at the permissive temperature degraded poly-ubiquitinated Sic1p efficiently, even at a higher temperature. Interestingly, their enzyme activity was even higher at a higher temperature, indicating that once made, mutant proteasomes are stable and have little defect in the proteolytic function. These results suggest that the deficiency in protein degradation observed in vivo be rather due to a defect in the assembly of a holoenzyme at the restrictive temperature. Indeed, both rpn6 mutants grown at the restrictive temperature were defective in assembling the 26S proteasome. A striking feature of the rpn6 mutants at the restrictive temperature was that there appeared a protein complex, composed of only four out of the nine lid components, Rpn5p, Rpn8p, Rpn9p and Rpn11p. Altogether, we conclude that Rpn6p is essential for the integrity/assembly of the lid, in the sense that it is necessary for the incorporation of Rpn3p, Rpn7p, Rpn12p and Sem1p (Rpn15p) into the lid, thereby playing an essential role in the proper function of the 26S proteasome.