The myb gene family consists of three members, the c-myb proto-oncogene and two myb-related genes (A-myb and B-myb), all of which encode nuclear DNA-binding proteins. Unlike c-myb, which plays a critical role in hematopoietic cells, B-myb is expressed in a large spectrum of hematopoietic as well as non-hematopoietic cells and has been implicated in the control of cell proliferation. The isolation of B-myb cDNA clones from several species has shown that B-myb shares limited homology to the so-called exon 9A of the c-myb gene. This exon is involved in differential splicing as only a subfraction of c-myb mRNA contains exon 9A sequences. The presence in the B-myb cDNA of a sequence related to the exon 9A of c-myb has prompted us to investigate whether B-myb mRNA is also spliced differentially. We here show that B-myb mRNAs containing or lacking exon 9A related sequences are present in many cell types. In contrast to c-myb, where RNA containing the exon 9A constitutes only a minor mRNA fraction, B-myb RNA containing the exon 9A related sequences is the major mRNA form. The proteins encoded by the two B-myb mRNA species are unable to activate promoters to which they bind. Curiously, both B-myb proteins differ in their ability to activate the HSP70 promoter by a myb binding-site independent mechanism; B-myb protein containing exon 9A related aminoacid sequences activates the HSP70 promoter much more potently than the B-myb protein which lacks these sequences. Our results suggest that differential splicing may be a general feature of the members of the myb family and provide first evidence for functional differences of the splice variants.