The B-Myb transcription factor has been implicated in coordinating the expression of genes involved in cell cycle regulation. Although it is expressed in a ubiquitous manner, its transcriptional activity is repressed until the G(1)-S phase of the cell cycle by an unknown mechanism. In this study we used biochemical and cell-based assays to demonstrate that the nuclear receptor corepressors N-CoR and SMRT interact with B-Myb. The significance of these B-Myb-corepressor interactions was confirmed by the finding that B-Myb mutants, which were unable to bind N-CoR, exhibited constitutive transcriptional activity. It has been shown previously that phosphorylation of B-Myb by cdk2/cyclin A enhances its transcriptional activity. We have now determined that phosphorylation by cdk2/cyclin A blocks the interaction between B-Myb and N-CoR and that mutation of the corepressor binding site within B-Myb bypasses the requirement for this phosphorylation event. Cumulatively, these findings suggest that the nuclear corepressors N-CoR and SMRT serve a previously unappreciated role as regulators of B-Myb transcriptional activity.