A straightforward method for the assembly of fluorescent polyrotaxanes is described here. The consecutive threading of N-(6(I)-desoxy-beta-cyclodextrin-6(I)-yl)-N'(5-fluoresceinyl)-thiourea, beta-CD-F, and alpha-CD onto poly(N,N-dimethyliminium-hexamethylen-N',N'-dimethyliminium-decamethylen e chloride.), I-6,10, leads to a fluorescent polyrotaxane. Free beta-CD-F could be distinguished from threaded beta-CD-F by gel electrophoresis. Since the dissociation of threaded alpha-CD rings is sterically hindered, the obtained polyrotaxanes are kinetically stable at 25 degrees C, and they did not require further stabilization by the attachment of stopper groups at the chain ends. Single polyrotaxane entities could be visualized with both fluorescence microscopy and atomic force microscopy. (C) 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6223-6230, 2009