Background. Protamine is commonly used in cardiac surgery to reverse the anticoagulant effects of heparin. We investigated the role of different nitric oxide synthase pathways in the response of the human internal thoracic artery to protamine and evaluated whether heparin could prevent this effect. Methods. A tension-recording method was used to obtain baseline measurements of contractions of human internal thoracic artery rings achieved with norepinephfine. Isolated internal thoracic artery rings were suspended in two organ chambers. One contained Krebs-Henseleit solution and served as control. The other contained a heparin or Nω-Nitro-L-arginine (L-NAM, an inhibitor of both endothelial and inducible nitric oxide synthase) or a specific inhibitor of inducible nitric oxide synthase, aminoguanidine. mine were added to both chambers and dose-response curves were obtained. Results. Protamine was found to relax contracted internal thoracic arteries 56% ± 4.7% of baseline measurements in a concentration-dependent manner. When LNAM was added, protamine caused only a slight decrease of tension. There were no differences in the relaxing effect of protamine in the presence of aminoguanidine or heparin. Conclusions. Protamine induces nitric oxide-dependent relaxation of the internal thoracic artery by activation of endothelial nitric oxide synthase pathway. Heparin could not prevent this relaxing effect of protamine.