The ionic strength-electrophoretic mobility relationship of human serum albumin was studied at pH 4.0 in acetate-NaCl buffer over a wide range of ionic strength values. A 5% albumin soln. which gave a single boundary at pH 8.6 in barbiturate buffer, 0.1 ionic strength, showed separation into a number of ascending boundaries at an ionic strength of 0.005 (barbiturate buffer). Similarly, at pH 4.0 in acetate-NaCl buffer, the number of ascending boundaries increases from 2 to 5 as the ionic strength is decreased from 0.2 to 0.02. Under th se conditions, the area under the fastest moving peak increases continuously at the expense of the slower-moving peak adjacent to it. Using an ionic strength of 0.03 (pH 4.0) as a standard condition, since it gives 4 clearly resolved boundaries, various other factors which may influence the electrophoretic pattern were studied, e.g., protein concn., method of prepn. of albumin fraction, time of run, and technique of dialysis used. Reproducible values for the areas under each of the 4 peaks were obtained at protein concns. between 1 to 5% and with mechanical dialysis for 5 hrs. or longer at 25[degree] against the acetate-NaCl buffer, 0.03 ionic strength. Preliminary work indicates that the electrophoretic sub-fractionation of other proteins or protein fractions, e.g., gamma globulin, is also possible and that this behavior may be part of a more generalized phenomena. The possible explanation of these phenomena await quantitative analysis of the components present under each observed peak. However, evidence is presented that boundaries probably represent protein components and not artefacts.