Reactive oxygen species were previously shown to trigger p21(Cip1) protein degradation through a proteasome-dependent pathway, however the detailed mechanism of degradation remains to be elucidated. In this report, we showed that p21(Cip1), was degraded at an early phase after low dose H2O2 treatment of a variety of cell types and that preincubation of cells with the antioxidant, N-acetylcysteine, prolonged p21(Cip1) half-life. A mutant p21(Cip1) in which all six lysines were changed to arginines was protected against H2O2 treatment. Direct interaction between P21(Cip1) and SkP2 was elevated in the H2O2-treated cells. Disruption of the two nuclear export signal (NES) sequences in p21(Cip1), or treatment with leptomycin B blocked H2O2-induced p21(Cip1) degradation. Altogether, these results demonstrate that reactive oxygen species induce p21(Cip1) degradation through an NES-, Skp2-, and ubiquitin-dependent pathway. (c) 2007 Elsevier Inc. All rights reserved.