MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21(Waf1). The present study was designed to characterize the mechanism(s) by which MDM2 induces p21(Waf1) degradation. We first determined the regions of MDM2 required for p21(Waf1) degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21(Waf1) interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21(Waf1) degradation. Specifically, amino acids 226-250 of MDM2 were required for p21(Waf1) binding and degradation, and amino acids 251-260 were necessary for p21(Waf1) degradation. The latter region induced a conformation change in p21(Waf1), increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226-250 or aa 251-260), its capacity to promote p21(Waf1) degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21(Waf1)) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties.