The migration behavior of nuclease A from Staphylococcus aureus and 11 of its variants in capillary zone electrophoresis (CZE) was investigated in the light of their three-dimensional structure known from X-ray crystallography and nuclear magnetic resonance (NMR) measurements. Nuclease A (molecular mass 16.8 kDa, pKa 10.3) and the variants differ only in a single amino acid residue and have a very similar crystal structure. With the use of coated quartz capillaries and suitable buffers, the protein migration was investigated at pH from 2.8 to 9.5 without interference by wall adsorption. Although the selectivity of the electrophoretic system for the proteins was mainly determined by their charge differences, certain variants having the same net charge could also be readily separated under nondenaturing conditions. For instance, the mobility of variant K116A was sufficiently higher than that of K116G so that they could be separated by CZE. The structures of both variants are the same except for the solvent-exposed loop containing residue 116. For this reason, the difference in electrophoretic mobilities can be attributed to the fact that in K116G the backbone of the 112 to 117 amino acids protrudes slightly from the protein, with a concomitant increase in the hydrodynamic radius with respect to that of K116A. Consequently, K116G shows a smaller mobility than K116A due to its larger hydrodynamic radius despite its smaller molecular mass. The interpretation of the experimentally measured mobilities of such closely related proteins therefore requires not only consideration of their electrostatic charge but also the fine details of their molecular structures.