Overexpression of p21(cip1) induces cell cycle arrest. Although this ability has been correlated with its nuclear localization, the evidence is not conclusive. The mutants that were used to inhibit its nuclear translocation could no longer bind to several proteins known to interact with the last 25 amino acids of p21(cip1). Here we used point mutation analysis and fusion of the proteins to DsRed to identify which amino acids are essential for the nuclear localization of p21(cip1). We conclude that amino acids RKR(140-142) are essential for nuclear translocation of p21(cip1). While wild-type DsRed-p21 induces cell cycle arrest in 95% of transfected cells, overexpression of cytoplasmatic p21AAA(140-142) arrested only 20% of transfected cells. We conclude that cytoplasmatic p21, with no deletion in the C-terminal region, had a much lower capacity to arrest the cell cycle.