In this report, we have prepared self-assembling nanospheres of hydrophobized pullulans. Pullulan acetate as a hydrophobized pullulan was synthesized by acetylation of pullulan and characterized by Fourier transform infrared (FTIR) measurement. From the results of photon correlation spectroscopy (PCS), hydrophobized pullulans could be self-assembled in water as nanospherical aggregates, and their number-average particle size was 74.3 +/- 38.2 nm with a unimodal distribution. Also, morphological studies observed by transmission electron microscopy (TEM) showed that self-assembly of hydrophobized pullulans results in nice spherical shapes with a size range of about 50-100 nm, which was in accordance with PCS measurements. Their size and morphology have acceptable properties for intravenous injectable drug-targeting carriers. The fluorescence probe technique was used for self-association of hydrophobized pullulans in water using pyrene as a hydrophobic probe. From the fluorescence measurement, the fluorescence intensity of pyrene increased with increasing concentration of hydrophobized pullulans, which indicates self-assembly formation of hydrophobized pullulans in water. Also, in the fluorescence excitation spectrum, a red shift was observed with increasing concentration of hydrophobized pullulans. These results also revealed that hydrophobized pullulans could be self-assembled in water, and from the plot of I337/I334 versus log c of hydrophobized pullulans, the critical association concentration was 0.0022 g/l, which was considerably lower than that of low molecular weight surfactants or poloxamer. A drug loading study was performed using clonazepam (CNZ) as a hydrophobic model drug. We observed that the higher the feeding amount of drug was, the more the drug loading contents were, the lower the drug loading efficiency was, and the larger the particle size was. CNZ was released from nanospheres via pseudo-zero-order kinetics, and the increased drug loading contents led to slower release of the drug.