Background. The clinical and experimental use of polyurethane heart valve prostheses has been compromised by thrombosis and calcified thrombus. This is caused in part by the lack of an intact endothelium on these implant surfaces. We hypothesize that endothelial seeding of a polyurethane heart valve leaflet with autologous sheep blood outgrowth endothelial cells (BOECs) could be achieved with cholesterol-modified polyurethane (PU-Chol) to promote BOEC adhesion, thereby resulting in an intact, shear-resistant endothelium that would promote resistance to thrombosis. Methods. Cholesterol-derivatized polyurethane was formulated by bromoalkylation of the urethane nitrogens followed by reactive attachment of mercaptocholesterol. In vitro shear flow studies were carried out comparing BOEC retention on control surfaces versus PU-Chol using forces comparable to those observed in vivo with cardiac valves (75 dyne/cm 2). Autologous sheep BOECs were seeded onto PU-Chol before pulmonary leaflet replacement surgery under cardiopulmonary bypass. Studies were terminated at 30 and 90 days followed by retrieval analyses. Results. Blood outgrowth endothelial cell seeding of PU-Chol surfaces resulted in an endothelial monolayer that was positive for von Willebrand factor. Polyurethane-cholesterol demonstrated significantly greater BOEC adhesion under 75 dyne/cm2 shear force in vitro than control polyurethane (75.3% ± 12.3% versus 5.8% ± 3.9%, respectively; p < 0.001). Sheep pulmonary cusp replacements demonstrated retention of seeded BOECs on PU-Chol leaflets with no significant differences in the extent of cellular density comparing unimplanted specimens with explants. Control explants (nonseeded PU-Chol and nonseeded polyurethane) demonstrated no evidence of endothelial recruitment. Conclusions. Polyurethane-cholesterol represents a polyurethane formulation with very high adhesive properties for BOECs under heart valve level shear forces both in vitro and in vivo.