We investigated the effects of compounds with two covalently linked netropsin moieties (bis-netropsin) on the function of mammalian type I DNA topoisomerase (topo I) in vitro. We initiated these studies because earlier studies had shown that certain bis-netropsins possess a several-fold higher antitumor and antiviral activity than netropsin. We confirmed that the parent compound netropsin, but not its bifunctional derivatives, induce supercoils in closed DNA. We determined that bis-netropsins inhibit the binding of topo I to DNA more efficiently than netropsin and that bis-netropsins but not netropsin induce specific DNA strand cleavage in the presence of topo I. We discuss a model explaining the different effects of netropsin and bis-netropsins on topo I.