Development of a small diameter prosthetic vascular graft with surface based antithrombin properties should aid in maintaining early graft patency in small vessel reconstruction. The purpose of this study was to bind covalently a basecoat protein (canine serum albumin [CSAJ) and a potent antithrombin agent (recombinant hirudin [rHir]) to 4 mm inner diameter poly(carbonate urea) urethane grafts with reactive carboxylic acid groups (cPU). 125I-CSA was covalently bound to 1 cm length segments of cPU grafts using the carbodimide cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To bind 125I-rHir covalently, CSA was modified with the heterobifunctional cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) before linkage to the cPU surface with EDC (cPU-CSA-SMCC). 125I-rHir was modified with Traut's reagent and reacted with the cPU-CSA-SMCC surface, covalently linking 125I-rHir to surface bound CSA. 125I-CSA binding to the cPU graft surface (34,235 ng/segment) was ninefold, sevenfold, and 10-fold greater than controls with nonspecifically bound 125I-CSA. Covalent linkage of 125I-rHir to the cPU-CSA-SMCC surface (9,974 ng/segment) was 172, 192, and 142-fold greater than controls with nonspecifically bound 125I-rHir. Surface antithrombin properties were characterized using a chromogenic assay to measure residual thrombin activity. Evaluation of surface antithrombin activity showed significantly greater 131I-thrombin inhibition and binding by the cPU surface with covalently bound 125I-rHir, as compared with controls. Release of 125I-rHir from the cPU surface was minimal as compared with controls. Therefore, rHir can be covalently linked to a novel small diameter polyurethane vascular graft surface while maintaining its potent antithrombin properties.