DNA topoisomerase IIalpha (topo IIalpha) is an essential proliferation-dependent nuclear enzyme which has been exploited as an anti-tumor drug target. Since the proliferative status of human leukemia cells is associated with expression of the c-myb proto-oncogene, c-Myb was investigated as a trans-activator of the topo IIalpha gene. Using topo IIalpha promoter-luciferase reporter plasmids, c-myb expression caused trans-activation of the topo IIalpha promoter a maximum of approximately 4.5-fold over basal levels in HL-60 human promyelocytic leukemia cells. Trans-activation was submaximal with higher levels of c-myb expression plasmid but a Myb protein lacking its negative regulatory domain resulted in approximately 19-fold trans-activation. Mutagenesis and 5'-deletion studies revealed that Myb trans-activation was mediated via a Myb-binding site at positions -16 to -11 and that this region governed the bulk of basal topo IIalpha promoter activity in human leukemia cells. Trans-activation of topo IIalpha by c-Myb was lymphoid- or myeloid-dependent. However, B-Myb, a more widely-expressed Myb family member, caused topo IIalpha trans-activation in both HL-60 cells and HeLa epithelial cervical carcinoma cells. These data provide evidence for a new Myb-responsive gene which is directly linked to and required for cellular proliferation.