Polyelectrolytes carrying cholesterol (Chol) pendants were synthesized by free-radical copolymerization of sodium 2-(acrylamido)-2-methylpropanesulfonate with cholesteryl methacrylate (CholMA) or cholesteryl 6-methacryloyloxyhexanoate (Chol-C-5-MA), with the contents of CholMA and Chol-C-5-MA units in the copolymers ranging from 0.5 to 1.0 and 0.5 to 10 mol %, respectively. In the CholMA copolymer, Chol is directly linked to the polymer main chain by an ester bond, whereas in the Chol-C-5-MA copolymer, Chol is linked via a pentamethylene spacer. The association behavior of these Chol-carrying polymers in aqueous solution was investigated by turbidimetry, H-1 NMR, fluorescence, size exclusion chromatography (SEC), static light scattering (SLS), quasielastic light scattering (QELS), and viscometry, with a focus on the effect of the spacer bond on the self-association of Chol pendants. For fluorescence studies, Chol-carrying polymers labeled with a pyrene (Py) or naphthalene (Np) moiety were employed. Fluorescence of molecular Py solubilized in the hydrophobic microdomains of the polymers was also examined. Pyrene fluorescence data indicated that the Chol pendants underwent self-association in aqueous solution even if their contents in the polymers were as low as 1 mol %. For the 5 mol % Chol-C-5-MA-containing polymer, a strong tendency for interpolymer association was indicated by nonradiative energy transfer from Np to Py chromophores labeled on separate polymer chains. Proton NMR data for the 5 mol % Chol-C-5-MA-containing polymer in D2O indicated highly restricted motions of Chol groups arising from the self-association of Chol groups. Taken together with SEC data, SLS and QELS data showed that the Chol-C-5-MA copolymers have a much stronger propensity for interpolymer association than the CholMA copolymers, with the pentamethylene spacer bond facilitating pendant Chol groups to undergo interpolymer association. On the basis of the characterization data, an intermolecularly bridged "flower-type" micelle model was proposed for the aggregates of the Chol-C-5-MA copolymers.