The interaction of histamine with beef lung heparin has been characterized by 'H and I3C NMR spectroscopy. IH chemical shifts provide evidence that histamine forms a complex with heparin both at low pD where 2-Osulfo-a-~-idopyranosyluronic acid (IdoA-2s) residues are in the carboxylic acid form and at higher pD where the carboxyl groups are deprotonated. At low pD, the chemical shift data are consistent with weak delocalized binding of the diprotonated form of histamine by the highly negatively charged polymer. However, as the pD is increased and the carboxylic acid groups are titrated, a binding region with a high affinity for diprotonated histamine is created. It is proposed that, in this binding site, the ammonium group of histamine is hydrogen-bonded to the carboxylate group of an IdoA-2S residue while the imidazolium ring is located in a region surrounded on three sides by the sulfamido group of a 2-deoxy-2-sulfamido-6.Osulfo-a-Dglucopyr (GlcNS03-6S) residue and the 2-Osulfate and carboxylate groups of a second IdoA-2S residue. Evidence for site specific binding includes displacement of chemical shift titration curves to lower pD and increased shielding of specific heparin protons from the imidazole ring current. Similar 'H NMR measurements on the heparin-imidazole and heparin-N-acetylhistamine systems indicate that the highly negatively charged binding site created by deprotonation of the carboxylic acid groups has a high affinity in general for positively charged imidazolium rings. Nuclear Overhauser enhancements obtained from two-dimensional NOESY spectra of heparin at pD 2.4 and 7.4 indicate that, when the carboxylic acid groups of heparin are titrated, its conformation changes by a small rotation around the (IdoA-2S)-( 1-+4)-(GlcNS03-6S) glycosidic bond. This study provides evidence for the first time of the site specific binding of a biological molecule by the repeating (IdoA-2S)-( 1+4)-(GlcNS03-6s) disaccharide unit of heparin.