The functional role of the cyclin-dependent kinase inhibitor (CDKI) p21 CIP1 in differentiation of human myelomonocytic leukemia cells (U937) exposed to low concentrations of the antimetabolite 1-β-D-arabinofuranosylcytosine (ara-C) was examined utilizing a cell line stably expressing a p21 CIP1 antisense construct. Continuous exposure to 50 nM ara-C led to marked induction of p21 CIP1 at 48–72 h in empty-vector control cells but not in their antisense-expressing counterparts (p21AS/F4 and B8). Such treatment induced expression of the myelomonocytic differentiation marker CD11b in ∼ 35 % of control cells, but no evidence of maturation was noted in antisense-expressing lines. However, antisense-expressing cells exposed to low concentrations of ara-C exhibited a reciprocal increase in apoptosis, manifested by the appearance of cells with classic morphologic features and hypodiploid quantities of DNA, reduced mitochondrial membrane potential (Δψ m ), an increase in cytochrome c release into the cytosol, cleavage/activation of procaspases-9 and −3, and degradation of PARP and p27 Kip1 . Whereas empty-vector control cells exposed to 50 nM ara-C exhibited a decline in Bcl-2 expression, dephosphorylation of pRb, and an initial accumulation in S-phase, antisense-expressing cells did not. However, c-Myc down-regulation induced by low concentrations of ara-C was, if anything, more complete in antisense-expressing cells. Exposure of control but not antisense-expressing cells to ara-C led to phosphorylation/activation of MAP kinase at 24 h; moreover, the specific MEK/MAP kinase inhibitor PD98059 enhanced low-dose ara-C-mediated apoptosis only in wild-type cells. Lastly, exposure to 50 nM ara-C for 72 h resulted in detectable levels of cytoplasmic p21 CIP1 , a phenomenon associated with resistance to apoptosis, only in empty vector controls. Collectively, these findings demonstrate a functional role for p21 CIP1 in leukemic cell maturation induced by low concentrations of ara-C. They also indicate that, as in the case of more conventional differentiation-inducers such as phorbol esters, disruption of the p21 CIP1 response after exposure to low concentrations of the cytotoxic drug ara-C prevents leukemic cells from engaging a maturation program, but instead directs them along an apoptotic pathway.