Electrospun scaffolds for in situ tissue engineering can be prepared with different fiber diameters to influence cell recruitment, adhesion, and differentiation. For cardiovascular applications, we investigated the impact of different fiber diameters (2, 5, 8, and 11 mu m) in electrospun poly(epsilon-caprolactone) scaffolds on endothelial colony forming cells (ECFCs) in comparison to mature endothelial cells (HUVECs). In 2D cultures and on 2 mu m fiber scaffolds, ECFC morphology and phenotype resemble those of HUVECs. When cultured on scaffolds with 5-11 mu m fibers, a different behavior was detected. HUVECs developed a cytoskeleton organized circumferentially around the fibers, with collagen alignment in the same direction. ECFCs, instead, aligned the cytoskeleton along the scaffold fiber axis and deposited a homogeneous layer of collagen over the fibers; moreover, a subpopulation of ECFCs gained the alpha SMA marker. These results showed that ECFCs do not behave like mature endothelial cells in a 3D fibrous environment.