The direct manipulation by atomic force microscopy (AFM) of individual macrocycles within a rotaxane offers a potential route to a new sequencing tool for complex macromolecules such as polysaccharides, glycoproteins and nucleic acids. In this paper we demonstrate for the first time that a sliding contact made between a macrocycle, a-cyclodextrin, and its polymer axle by an AFM tip can be used to map the positions of specific groups along the polymer as if they were beads along a string, thereby generating sequence information. We find very good agreement (linear fit with slope = 1.03, R2 = 0.968) between the calculated and measured positions of phenylenediamine and benzenetricarboxylic acid groups within polymers of polyethylene oxide (PEO). The rupture force profiles attributable to the dethreading interactions of phenylenediamine and benzenetricarboxylic acid differ observably from each other and, in the latter case, from the rupture of the corresponding host–guest complex. As well as opening the way to a macromolecular sequencing technique, the ability demonstrated by this method to manipulate the dethreading of a rotaxane offers a new tool for investigating the process energetics in a wide array of spontaneously forming and forced rotaxane systems.