The molecular weight and size of recombinant Hepatitis B surface antigen (HBsAg) derived from Chinese hamster ovary (CHO) and the Hansenula polymorph have been characterized by high-performance size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALLS). The average molecular weight of CHO-derived HBsAg particle (CHO-rHBsAg) (4921 kDa) was higher than that of H. polymorpha yeast strain (Hans-rHBsAg) (3010 kDa). The size of CHO-rHBsAg (22.1 nm) is nearly the same as that of native HBsAg compared to 18.1 nm for Hans-rHBsAg. The average monomer numbers were found to be 155 for CHO-rHBsAg and 86 for Hans-rHBsAg, respectively. The data obtained support the assumption that the higher immunogenicity of CHO-derived HBsAg is related to its more favorable macromolecular assembly structure.