Aclacinomycin A (aclarubicin) is an anthracycline anticancer agent with demonstrated activity against both leukemias and solid tumors. Previous results suggested that a major activity of aclacinomycin A is the inhibition of topoisomerase II catalytic activity. We have applied a yeast system to test whether aclacinomycin A is a topoisomerase II inhibitor in vivo and to test whether we could identify other important targets of this drug. We have found that overexpression of yeast topoisomerase II confers resistance to aclacinomycin A in yeast, consistent with the hypothesis that this drug is a catalytic inhibitor of topoisomerase II. Interestingly, we have also found that in yeast, aclacinomycin A, like camptothecin, stabilizes topoisomerase I cleavage. We carried out biochemical analysis with purified human topoisomerase I and demonstrated that this drug efficiently stabilizes topoisomerase I covalent complexes, indicating that aclacinomycin A represents a novel class of combined topoisomerase I/II inhibitor.