Human femoral arteries were cultured up to 56 days. Samples were processed for light, immunohistochemical, and transmission electron microscopy. Arteries became rapidly depopulated; at day 42, an endothelial lining (CD31(+), Weibel-Palade bodies) developed on the intima; endothelium was in continuity with mesenchymal stromal cells (CD44(+), CD90(low), CD105(low)) placed on adventitia. The media-adventitia area showed heterogeneous cell populations. In long-term organ culture, femoral artery develops a continuous cell coverage that differentiates to endothelium on the intima exclusively. This suggests that distinct topographical factors, such as resident progenitors and/or matrix signals, are able to regulate vascular homeostasis in adult life.