The suitability of using a fibroblast-seeded polymer scaffold as a substrate for forming an endothelial monolayer which is responsive to flow has been investigated. The results indicate that a confluent monolayer of endothelial cells can be formed on the top surface of the fibroblast/polymer substrate. This monolayer has an elongated, oriented morphology and alignment even in static culture, and the application of shear stress perpendicular to the endothelium's static alignment causes a progressive reorganization of the cytoskeletal component, F-actin, with a corresponding change in endothelial cell shape from elongated in the direction of flow, to a more "cobble-stone" morphology, to finally elongation in the direction of flow. The production of nitric oxide by this co-culture increases not only in a time and shear stress dependent fashion, but also as a function of the direction of flow in relation to the static alignment of the endothelium. The data obtained also indicate that the coculture had higher levels of ICAM-1 production and monocyte adhesion than seen in ECs on tissue culture plastic, which may be a function of the endothelial cells being in direct contact with the fibroblast cell line. These results demonstrate that a fibroblast/polymer scaffold can serve as a suitable substrate for ECs and that such a monolayer demonstrates the normal EC responsiveness to flow.