Targeting endothelial progenitor cells (EPC) for in vivo endothelialization is an emerging and promising approach for the development of cardiovascular medical devices. This study examined the efficacy of capturing CD34 positive EPC onto polyurethane (PU) immobilized with CD34 monoclonal antibodies (mAbs) (a biomecial polymer for cardiovascular devices). Electrospun PU matrices were fabricated and heparin was immobilized along with CD34 mAb. The modified PU surfaces at each step were characterized by contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). XPS showed that each surface was modified, as expected in terms of the chemical composition. The amine-terminated poly(ethylene glycol)(PEG)-PU surface was considerably more hydrophilic than the PU surface. In addition, its surface roughness was similar to the PU surface, indicating that PEG was sufficiently and evenly grafted onto the PU surface. The CD34 mAb-immobilized PEG-PU surface was less hydrophilic than PEG-PU and extremely rough as compared to the other two surfaces. These results demonstrate that relatively large CD34 mAbs were immobilized on the PU surface. The surface density of the immobilized CD34 mAb, which was quantified using an enzyme-linked immune-sorbent assay (ELISA), was increased to similar to 40 ng/cm(2) by varying the feed amount up to similar to 200 ng/cm(2) and co-immobilizing with heparin. These results suggest that the co-immobilization with heparin can provide two benefits: inhibiting initial occlusion and improving the surface density of CD34 mAb. The in vitro cell study also demonstrated that the CD34 mAb-immobilized PU surface was favorable for cell attachment and proliferation. Therefore, in this study, a novel approach was developed to achieve endothelialization for cardiovascular applications by immobilizing CD34 onto PU, and the synergistic effects of co-immobilization with heparin on the bioactivity of the PU surfaces was demonstrated.