The applicability of the membrane-impermeant protein cross-linker bis(sulfosuccinimidyl) suberate (B S) to the determination of membrane sidedness of proteins was tested in 3T3-L1 cells and in erythrocytes. Binding of BS3 to proteins was apparent in electrophoresis. In three proteins of 3T3-L1 cells, protein kinase-C epsilon, protein kinase-C zeta, and glyceraldehyde-3-phosphate dehydrogenase, BS3 action was detectable in SDS-PAGE with immunoblotting. This enabled confirmation of the well-known intracellular localization of these proteins. In cathepsin E of erythrocytes, a mobility increase in nondenaturing PAGE was the most prominent effect of BS3 treatment. A mechanism for the increase in mobility due to BS3 binding is suggested. Cathepsin E was found to be located at the intracellular side of the membrane, in accordance with existing evidence.