The effects of seeding density have often been overlooked in evaluating endothelial cell-biomaterial interactions. This study compared the cell attachment and proliferation characteristics of endothelial cells on modified poly (L: -lactic acid) (PLLA) films conjugated to gelatin and chitosan at low and high seeding densities (5,000 and 50,000 cells/cm(2)). During the early stage (2 h) of cell-biomaterial interaction, a low seeding density enabled us to observe the intrinsic surface-dependent differences in cell attachment capacity and morphogenesis, whereas extensive intercellular interactions at high seeding density masked differences between substrates and improved cell attachment on low-affinity substrates. During the later stage of cell-biomaterial interaction over 7-days of culture, the proliferation rate was found to be surface-dependent at low seeding density, whereas this surface-dependent difference was not apparent at high seeding density. It is recommended that low seeding density should be utilized for evaluating biomaterial applications where EC density is likely to be low, such as in situ endothelialization of blood-contacting devices.