We have synthesized and characterized N-hydroxysulfosuccinimide, a new hydrophilic ligand for the preparation of active esters. We have incorporated this ligand into two new protein cross-linking reagents, 3,3'-dithiobis(sulfosuccinimidyl propionate) and bis(sulfosuccinimidyl) suberate. In experiments with rabbit muscle aldolase, it is demonstrated that both of these reagents are highly efficient protein cross-linkers at physiological pH and that 3,3'-dithiobis(sulfosuccinimidyl propionate) is quantitatively cleavable by reduction under mild conditions. In experiments with intact human erythrocytes and erythrocyte membranes, it is shown that both reagents are membrane impermeant and that when erythrocytes are treated with either reagent, both cross-link subunits of the anion channel (band 3) to covalent dimers at the extracytoplasmic membrane face.