The self-aggregation behavior and microscopic characteristics of hydrophobically modified glycol chitosans (HGCs), prepared by covalent attachment of 5β-cholanic acid to glycol chitosan, were investigated by using 1H NMR, dynamic light scattering, fluorescence spectroscopy, and transmission electron microscopy (TEM). The HGCs formed self-aggregates in an aqueous phase by intra- or intermolecular association between hydrophobic 5β-cholanic acids attached to glycol chitosan. The critical aggregation concentrations (cacs) of the HGCs were dependent on the degree of substitution (DS) of 5β-cholanic acid and were significantly lower than those of low molecular weight surfactants. The mean diameters of the self-aggregates decreased with the increase in the DS of 5β-cholanic acid because of the formation of compact hydrophobic inner cores. The TEM images demonstrated that the shape of the self-aggregates, on the basis of the HGCs, is spherical. The partition equilibrium constants (Kv) of pyrene, measured in the ...