The interaction of Na +, Ca 2+, Mg 2+, Zn 2+ and La 3+ with heparin, a highly negatively charged glycosaminoglycan, was studied by 1H and 23Na nuclear magnetic resonance spectroscopy. 1H chemical shift and nuclear Overhauser effect (NOE) data indicate that the counter ions Na +, Ca 2+ and Mg 2+ interact with the low pH, carboxylic acid form of heparin by delocalized, long-range electrostatic interactions. At higher pH, 1H chemical shift and NOE data indicate that Na + and Mg 2+ continue to interact with heparin in the same manner, even upon deprotonation of the carboxylic acid group; however, there is a site-specific contribution to the binding of Ca 2+, Zn 2+ and La 3+ under these conditions. Acid dissociation constants for heparin carboxylic acid groups and heparin-metal binding constants were determined from the pH dependence of 1H chemical shifts and 23Na spin-lattice ( T 1) relaxation times. Equilibrium constants for exchange of M 2+ for heparin-bound Na + were obtained from 23Na T 1 data. The acid dissociation constants show a strong dependence on Na + concentration due to the polyelectrolyte character of heparin.