Valvular endothelial cells interact with interstitial cells in a complex hemodynamic and mechanical environment to maintain leaflet tissue integrity. The precise roles of each cell type are difficult to ascertain in a controlled manner in vivo. The objective of this study was to develop a three-dimensional aortic valve leaflet model, comprised of valvular endothelium and interstitial cells, and determine the cellular responses to imposed lumenal fluid flow. Two leaflet models were created using type I collagen hydrogels. Model 1 contained 1 million/mL porcine aortic valve interstitial cells (PAVICs). Model 2 added a seeding of the lumenal surface of Model 1 with approximately 50,000/cm(2) porcine aortic valve endothelial cells (PAVECs). Both leaflet models were exposed to 20 dynes/cm(2) steady shear for up to 96 h, with static constructs serving as controls. Endothelial cell alignment, matrix production, and cell phenotype were monitored. The results indicate that PAVECs align perpendicularly to flow similar to 2D culture. We report that PAVICs in model 1 express vimentin strongly and alpha-smooth-muscle actin (SMA) to a lesser extent, but SMA expression is increased by shear stress, particularly near the lumenal surface. Model 1 constructs increase in cell number, maintain protein levels, but lose glycosaminoglycans in response to shear. Co-culture with PAVECs (Model 2) modulates these responses in both static and flow environments, resulting in PAVIC phenotype that is more similar to the native condition. PAVECs stimulated a decrease in PAVIC proliferation, an increase in protein synthesis with shear stress, and reduced the loss of glycosaminoglycans with flow. Additionally, PAVECs stimulated PAVIC differentiation to a more quiescent phenotype, defined by reduced expression of SMA. These results suggest that valvular endothelial cells are necessary to properly regulate interstitial cell phenotype and matrix synthesis. Additionally, we show that tissue-engineered models can be used to discover and understand complex biomechanical relationships between cells that interact in vivo.