Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.