We examined the mechanism responsible for the degradation of p21, a negative regulator of the cell division cycle. We found that p21 proteolysis requires functional ubiquitin and Nedd8 systems. Ubiquitinylated forms of p21 and p21(K0), a p21 mutant missing all lysines, are detected in vivo and in vitro, showing that the presence of lysines is dispensable for p21 ubiquitinylation. Instead, the free amino group of the N-terminal methionine of p21 is a site for ubiquitinylation in vivo. Although wild-type p21 is more abundantly ubiquitinylated than p21(K0) mutant due to the presence of internal lysine residues, their rates of proteolysis are indistinguishable. These results demonstrate that proteasomal degradation of p21 is regulated by the ubiquitin pathway and suggest that the site of the ubiquitin chain is critical in making p21 a competent substrate for the proteasome.