For determining the position of a defined site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a specific sequence while preserving the superhelicity. This is done by first introducing a specific nick, labeling the DNA by limited nick translation and sealing the nick with ligase. The superhelicity is controlled by including ethidium in the ligation reaction. Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a specific site relative to the end loop of the superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a defined distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a superhelix.