Flow and the associated shear stress have been shown to play an active role in the regulation of the structure and function of endothelial cells (EC) in vitro. Although cultured EC subjected to flow exhibit an elongated morphology and a decreased cell growth rate rather like those observed in vivo, there are differences in morphology and growth rate, as well as other characteristics, between in vitro and in vivo EC. This suggests that flow is only one of the many factors affecting EC differentiation in vivo. In this study, a co-culture model system was designed, which includes smooth muscle cells (SMC), a matrix of collagen type I, and a confluent monolayer of EC, and this simplified model of the arterial wall was subjected to a steady, laminar shear stress of 10 and 30 dyn/cm2. Under non-flow conditions, EC exhibited an elongated shape, but with a random orientation. In response to flow, there was an alignment with the direction of flow. This alignment occurred more rapidly at 30 dyn/cm2 than at 10 dyn/cm2. The collagen matrix was found to be primordial in the maintenance of a quiescent endothelium, even in the absence of SMC and flow, suggesting the importance of an organized extracellular matrix (ECM) in the differentiation of cells in vivo.