Expression of the B-Myb transcription factor is upregulated during late G1 phase of the cell cycle by an E2F-dependent transcriptional mechanism. B-Myb is specifically phosphorylated during S phase, suggesting that a cyclin-dependent kinase (Cdk) regulates its activity. Consistent with this notion, the S phase-specific cyclin A/Cdk2 was found previously to enhance B-Myb transactivation activity in cotransfected cells. There is evidence that B-Myb is a direct physiological target for cyclin A/Cdk2. Data indicate that phosphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 activity may contribute to cell line-specific B-Myb function . Nuclear entry of B-Myb is dependent on multiple nuclear localization signals (NLS's). Mutagenesis of the putative NLS's of B-Myb has identified two separate NLS's, NLS1 and NLS2. Each of the two NLS's is essential for efficient nuclear targeting.