Abstract Introduction: Establishing thrombosis resistant surface is crucial to develop successful tissue engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anticoagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Methods: Porcine carotid arteries were obtained from a local abattoir. Cellular components of the arteries were completely removed with a newly developed method of decellularization. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulfate salt and heparin-EDC. Bound heparin contents were measured by a quantitative colorimetric assay of toludine blue staining. Anti-coagulation property of bound heparin was determined with a clotting time assay using both human and pig fresh blood. In vivo platelet deposition of the vessel was carried out with a baboon model of the femoral arteriovenous external shunt and 111Indium labeling of platelets. Results: The average content of heparin in treated vessels was 35.6 ± 11.6 mg/g tissue (ranging from 31 to 42 mg/g tissue), which represented 6.21 ± 2.03 UPS heparin/cm 2 tissue (ranging from 5 to 8 UPS/cm 2 tissue). The stability of heparin linkage was tested by incubating the heparin linked vessels either in PBS at 37°C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Furthermore, several vessels were stored in 70% alcohol for more than 2 years and the contents of bound heparin was not reduced. Standardized small pieces (3.3 × 3.3 mm 2) of non-heparin bound vessels were clotted in both human and pig fresh blood within 13 min, while all heparin bound vessels did not form clot during 1 hour observation. There were 1.38 ± 0.07 × 10 9, and 0.64 ± 0.11 × 10 9 baboon platelets deposited on the control and heparin linked vessels, respectively, at 60 min. Conclusions: These data demonstrate that covalent linkage of heparin provides an effective and stable anti-coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue engineered biological vascular grafts which could use for human coronary or low extremity artery bypasses.