We have previously shown that a synthetic peptide (dL) consisting of amino acids 1013-1056 of human alpha topoisomerase II adopted an alpha-helix structure and formed a stable dimer coiled-coil in solution [Frère, V., Sourgen. F., Monnot, M., Troalen, F. & Fermandjian, S. (1995) J. Biol. Chem. 270, 17502-17507]. Here we studied two peptides, dP and dLshort, which are related to dL but which have a double substitution Leu1026-->Pro, Leu1037-->Pro and a deletion of the 15 C-terminal residues, respectively. The peptides were studied for their ability to form alpha-helix structures, coiled coils, and to inhibit topoisomerase II activity. In combining circular dichroism spectra with AGADIR prediction for helix structures, we demonstrated that the dLshort peptide, like its parent dL peptide, adopts an alpha-helix structure and can autoassociate into coiled-coils, while dP is completely devoid of such properties. Remarkably, only the dL and dLshort peptides act as good inhibitors of topoisomerase II in various in vitro assays. However, the dLshort peptide has a stronger helix potential and behaves as a much more potent inhibitor (5 microM versus 200 microM) compared to the dL peptide. All these data strongly suggest that the greater inhibitory effect demonstrated by the dLshort peptide is related to its higher ability to form a stable amphiphilic helix, which in turn better recognizes its homologous helical segment in topoisomerase II. Finally, we propose that the dL and the dLshort peptides could interfere with the enzymatic activity of topoisomersase II in modifying its autoassociation or translocation properties. Such peptides may serve as useful models for developing simpler and more specific inhibitors of topoisomerase II.