Camptosar (CPT-11) and Hycamtin (topotecan, TPT), are two clinically useful anticancer drugs of the camptothecin family which function by inhibiting human DNA topoisomerase I (TopoI). Successful inhibition of TopoI by camptothecins is known from structure-activity studies to require an intact lactone ring (ring E) functionality. Unfortunately, this lactone moiety is subject to hydrolysis under physiological conditions (i.e., at pH 7 and above) with each camptothecin agent existing in equilibria with its corresponding ring-opened carboxylate form. Our studies demonstrate that the positively charged and water-soluble TPT and CPT-11 congeners, as well as uncharged camptothecin, are capable of interacting directly with double-stranded DNA (dsDNA). Moreover, our results indicate that the dsDNA interactions of the camptothecin drugs of interest result in a marked stabilization of their active lactone forms. The presence of dsDNA, in fact, was found to promote the conversion of inactive carboxylate to active lactone. Our results thus provide the first evidence that duplex DNA devoid of TopoI may play a functional role in the biological activities of the camptothecins through the promotion of active lactone levels within the cell nucleus. These results suggest that the agents, upon reaching chromosomal DNA, may interact directly with DNA prior to the action by TopoI (although the site of drug binding to DNA is not necessarily at the site of topoisomerase I action). The DNA-associated drugs are likely to be in their active lactone forms and ready for the subsequent drug-DNA-enzyme complex formation.