The theory of chromatofocusing has been clarified, in a qualitative manner, in terms of the pH gradient. Donnan potential, non-uniformity of flow, retardation factor and the titration curve of the protein. The main equations were refined to include the effect of the moving frame. A number of representative experimental results from the literature are given to illustrate the utility and limitations of the method. Some of these data were used to substantiate the theory. It is shown that no displacement by ampholyte components can occur under the conditions of chromatofocusing.