Nuclear entry of the B-myb gene product (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. NLS2 contains two interdependent basic domains separated by 8 intervening spacer amino acids, and both basic domains are required for nuclear entry. Thus, NLS2 belongs to a class of bipartite NLS's. Like the NLS's in yeast transcription factor SW15, NLS2 contains a putative cdc2 kinase site. However, unlike the case of SW15, phosphorylation at this site did not affect the nuclear targeting of B-Myb.