A systematic protocol to fabricate self-assembly deoxycholate-chitosan nanospheres (DC-CsNS) by gamma-ray pre-irradiation and chemical modification was studied. Hydrophobic deoxycholic acid moieties were chemically conjugated to pre-irradiated chitosan. The influences of chitosan physical forms (i.e., colloid and flake) during irradiation, radiation doses, and the reaction system (heterogeneous or homogeneous) on the chemical modification and the particle shape and size were investigated. Pre-irradiation of chitosan in colloidal form produced smaller DC-CsNS particle size than that of flake form. In the heterogeneous reaction, the pre-irradiated dose influenced the DC-CsNS particle size, whereas in the homogeneous reaction all pre-irradiation doses gave an identical average size range of 30-50 nm. By pre-irradiation (10 kGy) of chitosan in colloidal form before heterogeneous chemical conjugation, it is possible to obtain DC-CsNS with an average size of 46 nm. DC-CsNS of about 50 nm in size could also be synthesized using homogeneous chemical conjugation onto non-irradiated chitosan with the addition of N-hydroxysuccinimide (NHS). (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci 123: 3309-3320, 2012