Chicken liver dihydrofolate reductase purified by Aga rose-methotrexate (MTX-Agarose) column has been analyzed. The boundsubstrates, dihydrofolate or folate, were removed by flat-bed isoelectric focusing electrophoresis. The effect of the bound-substrates on studying the conforrnation of the enzyme was discussed, and the wrong conclusion in the literature obtained with the enzyme in the presence of bound-substrates was pointed out.