Estrogens regulate cell proliferation in target tissues, including breast cancer by stimulating G(1)-S phase transition. Activation of cyclin E.Cdk2 through abrogation of the ability of p21(WAF1/Cip1) to bind to and inhibit cyclin-CDKs is a pivotal event in this process in MCF-7 breast cancer cells. A proposed mechanism is p21 sequestration into cyclin D1.Cdk4/6 complexes driven by estrogen-induced transcriptional activation of cyclin D1 gene expression. However, we now show that some E(2)-induced cyclin E.Cdk2 activation occurs in the absence of increased cyclin D1 levels and requires decreased p21 protein synthesis. Both mechanisms operate in the absence of major changes in total p21 protein levels and instead target a low abundance subset of newly synthesized p21. E(2)-induced activation of cyclin E.Cdk2 is mimicked by targeted inhibition of nascent p21 expression by antisense p21 oligonucleotides. Cyclin E.Cdk2 activation is completely inhibited by a combination of antisense cyclin D1 oligonucleotide transfection and elimination of the decrease in nascent p21 by infection with adenoviral-p21. These findings strongly support a central role for p21 in the early phase of E(2)-induced mitogenesis and highlight a major functional role for newly synthesized CDK inhibitory proteins.