The organization of cells into epithelium depends on cell interaction with both the extracellular matrix (ECM) and adjacent cells. The role of cell-cell adhesion in the regulation of epithelial topology is well-described. ECM is better known to promote cell migration and provide a structural scaffold for cell anchoring, but its contribution to multicellular morphogenesis is less well-understood. We developed a minimal model system to investigate how ECM affects the spatial organization of intercellular junctions. Fibronectin micropatterns were used to constrain the location of cell-ECM adhesion. We found that ECM affects the degree of stability of intercellular junction positioning and the magnitude of intra-and intercellular forces. Intercellular junctions were permanently displaced, and experienced large perpendicular tensional forces as long as they were positioned close to ECM. They remained stable solely in regions deprived of ECM, where they were submitted to lower tensional forces. The heterogeneity of the spatial organization of ECM induced anisotropic distribution of mechanical constraints in cells, which seemed to adapt their position to minimize both intra-and intercellular forces. These results uncover a morphogenetic role for ECM in the mechanical regulation of cells and intercellular junction positioning.pithelial sheets lie on a layer of extracellular matrix (ECM), the so-called basement membrane. In such epithelia, cells establish integrin-based adhesions on the basal part of the cell in contact with ECM, and cadherin-based intercellular adhesions on the apical part of contacting lateral domains, away from contact with ECM. The two adhesion systems display nonoverlapping spatial distributions. Both cell-cell and cell-ECM adhesions are required to establish proper epithelium morphology (1). They both participate in mechano-transduction of external physical cues into intracellular signaling (2). The biochemical nature of adhesion molecules engaged in intercellular adhesion, the energy of the interaction, as well as the mechanical tension developed along intercellular junctions have been shown to govern epithelial cell shape and orient intercellular junctions in various systems (3-6). However, whereas the contribution of cell-cell adhesion to epithelial topology has been the focus of many studies, much less attention has been paid to the role of ECM. ECM is a dynamic scaffold that is actively remodeled during morphogenesis, where it plays major roles in stimulating and guiding cell migration as well as orienting stem cell fate (7,8). ECM is also known to impart morphoregulatory signals to epithelia, and thereby regulates tissue morphogenesis (8, 9). However, the mechanism by which ECM guides cell positioning at the single-cell scale is still not known. ECM geometry has been shown to regulate intracellular architecture (10) and provide spatial information for cell polarization (1,11,12), but how it regulates cell positioning and thereby spatially organizes multicellular architectures remained to be i...