The separation of pancreatic trypsinogen and alpha -chymochypsinogen A by displacement chromatography was tested on a bifunctional adsorbent containing both butyl and carboxymethyl groups. Methacrylic triblock copolymer was synthesized and used as the displacer. Compared to the displacement results on commercial Butyl-Sepharose, it was found that both the separation and recovery of trypsinogen and alpha -chymochypsinogen A were improved. Adsorption isotherms of proteins were measured on both the commercial Butyl-Sepharose and the synthesized bifunctional adsorbents. It was found that the improvement of protein separation on bifunctional adsorbents was attributed to the alteration of the adsorption of trypsinogen. Charge repulsion between trypsinogen and the negatively charged carboxymethyl groups may account for the alteration. In addition, taking advantage of the effect of charge repulsion, the column regeneration became much easier on the column packed with bifunctional adsorbents.