Background: Only a few antitumor drugs inhibit the DNA breakage-reunion reaction catalyzed by topoisomerase. One is the camptothecin derivative topotecan that has recently been used clinically. Others are the glycosylated antibiotic rebeccamycin and its synthetic analog NB-506, which is presently in phase I of clinical trials. Unlike the camptothecins, rebeccamycin-type compounds bind to DNA. We set out to elucidate the molecular basis of their interaction with duplex DNA, with particular emphasis on the role of the carbohydrate residue. Results: We compared the DNA-binding and topoisomerase-I-inhibition activities of two isomers of rebeccamycin that contain a galactose residue attached to the indolocarbazole chromophore via an α (axial) or a β (equatorial) glycosidic linkage. The modification of the stereochemistry of the chromophore-sugar linkage results in a marked change of the DNA-binding and topoisomerase I poisoning activities. The inverted configuration at the C-1' of the carbohydrate residue abolishes intercalative binding of the drug to DNA thereby drastically reducing the binding affinity. Consequently, the α isomer has lost the capacity to induce topoisomerase-I-mediated cleavage of DNA. Comparison with the aglycone allowed us to determine the energetic contribution of the sugar residue. Conclusions: The optimal interaction of rebeccamycin analogs with DNA is controlled to a large extent by the stereochemistry of the sugar residue. The results clarify the role of carbohydrates in stereospecific drug-DNA interactions and provide valuable information for the rational design of new rebeccamycin-type antitumor agents.