The process of terminal differentiation is associated with exit from the cell cycle and loss of the proliferative potential of cells. The cyclin-dependent kinase inhibitors (CDIs) play critical roles in check-point functions during the cell cycle and as inhibitors of cell proliferation. Loss of their activities can impair development and differentiation and contribute to the uncontrolled proliferation characteristic of cancer cells. When the promyelocytic leukemia cell line HL60 is induced to differentiate in vitro, by a variety of agents, cellular levels of the CD1 proteins p21Cip1 and p27Kip1 are increased. To further address the roles of these two proteins in differentiation, we have overexpressed either a human p21Cip1 or p27Kip1 construct in HL60 cells. The overexpression of p21Cip1 accelerated both the monocytic and granulocytic differentiation of HL60 cells triggered by TPA or DMSO, respectively. The accelerated and more dramatic induction of differentiation seen in the p21Cip1 overexpressors was associated with a more rapid reduction of CDK2 kinase-associated activity, increased levels and more rapid dephosphorylation of the Rb protein, and increased levels of the cyclin D3 protein. Stable overexpression of p27Kip1 also enhanced TPA-induced differentiation of HL60 cells. These studies provide direct evidence that the increased expression of p21Cip1 and p27Kip1 play a causal role in the process of terminal differentiation of HL60 cells. Therefore, agents that enhance the expression of one or both of these proteins might be useful in therapy by enhancing the terminal differentiation of leukemia cells.