The sequence requirement for N-terminal cleavage and the proteasomal degradation of p27Kip1 and their relationship was investigated. Residues 5-8 were required for the cleavage and the mutation of S10 to E inhibited the cleavage. The C-terminal PEST sequence was necessary for the degradation and residue R165 was found to play an important role in the degradation. The inhibition of the cleavage by deleting residues 5-8 inhibited the degradation, while the fragment mimicking the cleavage product accelerated the degradation. Both the cleavage and degradation demonstrated a similar sensitivity toward proteasome inhibitors and ATP depletion. These two processes are thus suggested to be tightly linked and sequential. © 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.