Previous studies have shown that B-Myb, a conserved member of the Myb transcription factor family, is a potent activator of the promoter of the human HSP70 gene but does not activate promoters containing Myb binding sites. We have now investigated the transactivation properties of B-Myb in more detail. We here report that B-Myb activates the HSP70 promoter by a novel mechanism which involves the heat shock element (HSE). Deletion analysis of B-Myb shows that a specific domain in the center of B-Myb, but not the DNA-binding domain is required for HSE-dependent transactivation. We also show that deletion of the C-terminal domain of B-Myb does not affect HSE-dependent transactivation but allows the protein to activate a promoter containing Myb binding sites. This suggests that the ability to activate Myb binding site containing promoters is repressed in the context of full length B-Myb and that HSE dependent and Myb binding site dependent transactivation are distinct functions of B-Myb. Finally, we report that cyclin D1 like B-Myb strongly activates the HSP70 promoter via the HSE. HSE-dependent transactivation is a novel activity of cyclin D1 and appears to be independent of the phosphorylation of the Rb protein. Our results reveal an interesting and unexpected connection between HSE-dependent gene activation and proteins expressed during the G1/S-transition of the cell cycle.