Ultra-fine particles of alpha-chymotrypsin were produced with a supercritical anti-solvent (SAS) technique by using dimethyl sulfoxide (DMSO) as a solvent and carbon dioxide as an anti-solvent. According to the vapor-liquid equilibrium (VLE) phase diagram of DMSO +carbon dioxide, the precipitation experiments have been conducted in different phase regions, including supercritical, superheated vapor, and vapor-liquid phase coexistence zones. It was found that the phase behavior of the mixtures during precipitation governed the product's morphology. Uniform networked nano-particles were obtained as the precipitation was implemented in the supercritical region. The uniformity of the resulting products became worse when the particles were precipitated around the critical region. Irregular micro-scale aggregated particles were formed in the superheated region, while both dense cake and spherical clusters were produced in the vapor-liquid coexistence region. A continuous stirred tank reactor (CSTR) model was applied to calculate the dynamic composition variations of mixtures in precipitator during the particle formation process. The crystalline and thermal property of the original and the SAS processed samples were analyzed by a wide-angle X-ray scattering (WAXS), a differential scanning calorimeter (DSC) and a thermogravimetric analyzer (TGA), respectively. No significant changes were found after SAS treatment. Besides, the biological activity of the SAS processed (x-chymotrypsin was retained in a range of 30-75% of the original protein. (c) 2007 Elsevier B.V. All rights reserved.