An indolocarbazole compound, NB-506, inhibits the activity of topoisomerase I by stabilizing the DNA-topoisomerase I complex (cleavable complex). NB-506 inhibited the religation step of topoisomerase I activity more potently than camptothecin or its derivative, topotecan. A cleavage assay using an end-labeled fragment of DNA revealed that the pattern of cleavage induced by NB-506 was different from that induced by camptothecin. The preferred cleavage sites of NB-506 were found to be not only T but also A or C at the 3'-terminus of the cleaved DNA (position -1), while the DNA cleavage sites of camptothecin always had T at position -1. At the 5'-terminus of the cleaved DNA (position +1), NB-506 showed a preference for G, which is a feature shared in common with camptothecin. Therefore, the difference in cleavage patterns was most likely due mainly to the preferred base at position -1. Moreover, the re-ligation rate was significantly slower at NB-506-selective sites, which had C at position-1, than at camptothecin-selective sites or at sites cleaved by both NB-506 and camptothecin. Our data suggest that NB-506 is an unique topoisomerase I poison and that its potent inhibition of topoisomerase I is partly dependent on retardation of re-ligation at sites selectively induced by NB-506.