Chitosan derivatives containing 2.8−5.1 deoxycholic acid groups per 100 anhydroglucosamine units of chitosan were synthesized by an EDC-mediated coupling reaction. Physicochemical properties of self-aggregates of the hydrophobically modified chitosans in aqueous media were studied by the dynamic light scattering method and fluorescence spectroscopy. The mean diameter (d_H) of DC4.2 self-aggregates in phosphate-buffered saline (PBS) solution (pH 7.2) was 159 nm with unimodal size distribution (variance = 0.082). It seemed that the interparticle interaction between self-aggregates was almost negligible. The mean diameter decreased with increasing degree of substitution (DS), pH, or ionic strength of the medium. The critical aggregation concentration (cac) of self-aggregates was determined from the fluorescence emission spectra of pyrene. The cac value of DC4.2 in PBS solution (pH 7.2) was 2.6 × 10^-2 mg/mL. The cac values decreased with increasing DS, pH, or ionic strength of the medium.