This study examines the effect of electrospun polyethylene terephthalate mats fiber diameter, orientation, and surface properties on the Human Aortic Endothelial Cell behavior. Mats with two different average fiber diameters (740 +/- 200 nm and 1.8 +/- 0.2 microm); orientations (low and high); NaOH-treated and untreated were prepared. NaOH treatment altered mats physical properties. AlamarBlue assay revealed that all four test mats supported cell adhesion and growth. Cell growth was observed to be faster for mat with large fiber diameter than for the small fiber diameter mat. Fluorescent staining and scanning electron microscopy showed that fiber diameter and orientation influenced cell morphology. Cells were randomly spread on the 740-nm diameter fibers whereas most of them were oriented along the fibers with 1.8 microm diameter. Mat with higher fiber alignment showed higher cell orientation. Cells penetrated into the mats having 1.8 +/- 0.2 microm fiber diameter but remained on the surface of the mat with 740 +/- 200 nm, as determined from histological analysis. These findings highly suggest that the two mats may be potential materials to construct a two layer vascular graft scaffold in which the mat with small diameter fibers forms the luminal surface and the mat with larger fiber diameter the abluminal surface.