Hepatitis B surface antigen (HBsAg) is known to adsorb to aluminum hydroxide adjuvant (AH) by ligand exchange between its accessible phosphate groups and surface hydroxyl groups of the adjuvant. To study the effect of the binding strength, five vaccines were prepared with AH or four samples of AH that were modified by pretreatment with different concentrations of potassium dihydrogen phosphate. The adsorptive coefficients ranged from 3660 to 250mL/mg based on the Langmuir adsorption isotherm and degrees of elution ranged from 1 to 31% when the vaccines were exposed to interstitial fluid in vitro. When tested in mice the four vaccines containing phosphate-treated AH (PTAH) induced significantly greater antibody responses than the vaccine containing AH, which had the highest adsorptive coefficient and the smallest degree of elution of HBsAg. The results indicated that antibody production is reduced when the antigen is adsorbed too strongly. Thus, the strength of adsorption of the antigen to an aluminum-containing adjuvant can affect the immunogenicity of the vaccine and should be optimized during vaccine formulation.