Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a co-culture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1 integrin complex, whereas ECs used either α5β1 or αVβ3 to spread on fibronectin (FN) adsorbed to plastic. ECs in co-culture lacked focal adhesions, but EC α5β1 bound to fibrillar FN on the SMC surface promoting rapid fibrillar adhesion formation. As assessed by both Western blotting and quantitative real-time RT-PCR, co-culture suppressed the expression of focal adhesion proteins and mRNA, while tensin protein and mRNA expression was elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in co-culture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in co-culture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.