Estrogen rapidly induces expression of the proto-oncogene c-myc. c-Myc is required for estrogen-stimulated proliferation of breast cancer cells, and deregulated c-Myc expression has been implicated in antiestrogen resistance. In this report, we investigate the mechanism(s) by which c-Myc mediates estrogen-stimulated proliferation and contributes to cell cycle progression in the presence of antiestrogen. The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Using stable MCF-7 derivatives with inducible c-Myc expression, we demonstrated that in antiestrogen-treated cells, the elevated mRNA and protein levels of p21(WAF1/CIP1), a cell cycle inhibitor, decreased upon either c-Myc induction or estrogen treatment. Expression of p21 blocked c-Myc-mediated cell cycle progression in the presence of antiestrogen, suggesting that the decrease in p21 is necessary for this process. Using RNA interference to suppress c-Myc expression, we further established that c-Myc is required for estrogen-mediated decreases in p21(WAF1/CIP1). Finally, we observed that neither c-Myc nor p21(WAF1/CIP1) is regulated by estrogen or antiestrogen in an antiestrogen-resistant MCF-7 derivative. The p21 levels in the antiestrogen-resistant cells increased when c-Myc expression was suppressed, suggesting that loss of p21 regulation was a consequence of constitutive c-Myc expression. Together, these studies implicate p21(WAF1/CIP1) as an important target of c-Myc in breast cancer cells and provide a link between estrogen, c-Myc, and the cell cycle machinery. They further suggest that aberrant c-Myc expression, which is frequently observed in human breast cancers, can contribute to antiestrogen resistance by altering p21(WAF1/CIP1) regulation.