Abstract A theory is formulated for an isoelectric focusing procedure which permits determination of intrinsic ligand-binding constants. The protein is first focused in the absence of ligand, after which ligand is added to the appropriate electrode compartment and then driven by the electric field into the focusing column where it complexes with the protein. The band of protein and its complexes moves to the constituent isoelectric point. An equation linearly relates the reciprocal of the overall distance moved to the reciprocal of the local concentration of ligand. The quotient of the intercept and slope gives the intrinsic binding constant. If the concentration of ligand in the electrode compartment is used in lieu of the local concentration, an apparent constant is obtained. Extrapolation of the apparent constant to infinite dilution of protein gives the intrinsic constant. For certain systems, conditions may be realized which give an apparent constant within 4% of the intrinsic constant.