Stable DNA binding by the mammalian CCAAT displacement protein (CDP)/Cux transcription factor was previously found to be up-regulated at the G(1)/S transition as the result of two events, dephosphorylation by the Cdc25A phosphatase and proteolytic processing, to generate an amino-truncated isoform of 110 kDa. In S phase, CDP/Cux was shown to interact with and repress the core promoter of the p21(WAF1) gene. Here we demonstrate that DNA binding by p110 CDP/Cux is down-modulated as cells progress into G(2). Accordingly, cyclin A-Cdk1 was found to bind to CDP/Cux and modulate its DNA binding activity in vitro and in vivo. Interaction with CDP/Cux required the presence of both cyclin A and a cyclin-dependent kinase (Cdk)-activating kinase-activated Cdk1 and involved the Cut homeodomain and a downstream Cy motif. Phosphorylation of serines 1237 and 1270 caused inhibition of DNA binding in vitro. In cotransfection studies, cyclin A-Cdk1 inhibited CDP/Cux stable DNA binding and prevented repression of the p21(WAF1) reporter. In contrast, mutant CDP/Cux proteins in which serines 1237 and 1270 were replaced with alanines were not affected by cyclin A-Cdk1. In summary, our results suggest that the phosphorylation of CDP/Cux by cyclin A-Cdk1 contributes to down-modulate CDP/Cux activity as cells progress into the G(2) phase of the cell cycle.