Three new dimeric analogues of ethidium cation in which the monomeric moieties are linked at the 3' positions by alpha,omega-diethers of varying length and composition have been synthesized. The circular dichroism spectra of all three compounds indicate that they double intercalate, and their effects on the thermal helix-coil transition profile of poly(dA-dT) show extremely high affinity for helical DNA, with details of the binding interaction depending on the length and composition of the connecting chain. The ability of the compounds to inhibit nucleic acid synthesis in L1210 cell culture also differed significantly, as did their antitumor effects against P388 leukemia and B16 melanoma. Compound 2, with 10 methylene groups in the connecting chain, is 5-20 times as potent as ethidium against murine P388 leukemia. These results clearly illustrate the advantage gained by incorporating a weak antitumor agent in a double-intercalating analogue.