The authors previously demonstrated that heparin immobilized surfaces showed excellent nonthrombogenic properties for extracorporeal membrane oxygenation experiments as long as 168 hr. The characteristics of the heparin immobilized surfaces include high heparin bioactivity and prevention of platelet adhesion and complement activation. However, it is not known whether the heparin immobilized surfaces would be effective for in vivo long-term implantation. Heparin bioactivity may be lost because of complete degradation or blocking of binding sites on heparin by adsorbed proteins. This study attempted to elucidate the in vivo long-term fate of heparin immobilized surfaces. The blood contacting surfaces of the ventricular assist device (VAD) made from polyurethane was modified with heparin immobilization and evaluated in a long-term sheep left VAD (LVAD) model for as long as 3 months. After removal of the VAD, heparin bioactivity was measured by Factor Xa assay. The blood contacting surfaces were analyzed with a scanning electron microscope, and the adsorbed proteins on the surfaces of the diaphragm were analyzed by SDS-PAGE and Western blotting. The thickness of adsorbed proteins on the surfaces also was measured by a confocal laser microscope. For the control ventricular assist devices, thrombus formation was observed within 1 month, whereas heparin immobilized VADs were able to operate thrombus free for periods as long as 3 months. The control surfaces demonstrated a thick adsorbed protein layer on thin surfaces, whereas heparin immobilized surfaces maintained thinner adsorbed proteins on thin surfaces. Anti Factor Xa activity of the heparinized surfaces disappeared after 15 days, but the surfaces remained nonthrombogenic even after heparin bioactivity was completely lost. The protein composition analyzed by SDS-PAGE showed an albumin dominant pattern on the heparinized surfaces. The band of 110 kD corresponding to C3b was detected only on the control surfaces, which possibly activated complement, and subsequently activated platelets and coagulation. Immunoblot showed degradation products of fibronectin and vitronectin on the control surfaces, which probably were promoted by surface generated protease, whereas the heparinized surfaces showed minimal degradation throughout the experimental periods. These results suggest that the heparin moiety has an ability to control adsorbed proteins, thereby inhibiting thrombus formation during in vivo long-term implantation.