c-myb is a proto-oncogene encoding a transcription factor which is highly expressed in hematopoietic progenitor cells. It regulates the expression of genes important for lineage determination, cell proliferation, and differentiation. Deregulation of c-myb expression is known to be involved in the development of human tumors, especially certain types of leukemia and breast and colon cancer. The c-Myb protein has thus been identified as an interesting therapeutic target. We recently discovered that some sesquiterpene lactones suppress Myb-dependent gene expression which is a new mechanism for these natural products' potential anti-cancer activity. We developed a test system to screen compounds for inhibitory activity on Myb-inducible reporter gene activation. Using this system we have now investigated 60 sesquiterpene lactones for their capacity to inhibit c-Myb-dependent gene activation. The IC50 values were in a range between 0.7 and >30 μM. The furanoheliangolide goyazensolide and the pseudoguaianolide helenalin acetate (IC50 = 0.6 and 0.7 μM, respectively) represent the most active inhibitors of c-Myb dependent gene expression found up to present. Control measurements for cell viability (MTS assay) proved that the observed activity on c-Myb dependent gene expression is not a function of cytotoxicity/unspecific cell damage. Structure-activity relationships were investigated by a QSAR approach based on flexible alignment of the most active compounds and a common pharmacophore model. These investigations resulted in a QSAR model which indicates that the potency of inhibitory activity on c-Myb-dependent transcription does not only depend on the presence of reactive Michael-acceptor features but also on their optimal spatial arrangement in the molecule.