Endothelial cell (EC) formation and distribution of both actin stress fibers and focal contacts on hydrophilic plasma polymers derived from gamma-butyrolactone (GBL) and n-vinylpyrrolidone (NVP) were examined to determine their ability to support endothelial cell growth in comparison to fibronectin. One hour after seeding, cells adhered and spread moderately on fibronectin with the development of defined actin stress fibers and focal adhesions compared to NVP and GBL, on which the cells were spread with poorly developed stress fibers and a perinuclear localization of vinculin. At 3 h, cells continue to spread more on fibronectin and NVP than GBL, and the cells on fibronectin had well-defined stress fibers terminating with sharp spikes of vinculin, typical of focal adhesions. At this time point, paxillin, a signaling component of focal adhesion complex, was predominantly localized at the focal contacts for well-spread EC on fibronectin and NVP, whereas it was almost entirely concentrated in the perinuclear region of less-spread cells on GBL. However, by 24h, cells were much more spread on all three surfaces with defined stress fibers and focal contacts although EC expression of vinculin and paxillin was moderate on GBL compared to fibronectin and NVP. These results suggest that EC can form cytoskeletal structures necessary for cell survival on plasma polymers, especially on more hydrophilic NVP, which could be exploited as interface material for seeding endothelial cells.