Abstract: A novel pH buffering system which is functionally stable under electrophoretic conditions employs buffering components in complementary buffer pairs. The buffering components are selected from among simple chemically defined ampholytes, weak acids and weak bases, and are paired together on the basis of their dissociation characteristics to define narrow overlapping buffering zones in which the buffering components exhibit low net electrophoretic mobility combined with desired pH buffering capacity. The narrow buffering zones cover from 0.4 to 1.25 pH units within a broad range of pH values of from about pH 3 to about pH 10. Specific buffering pH values within the narrow buffering zones are obtainable by varying the molar ratios of the two buffering components of the selected buffer pair. The buffering system may be used to form functionally stable precast narrow pH zone gradients in free solution, and has particular utility in isoelectric focusing and other electrophoretic processes.