When cultured in vitro cardiomyocytes lose their native highly organized structure and adopt a random orientation, potentially compromising many of their properties. In this study, we first engineered cardiomyocyte monolayers with an organized structure, endeavoring to mimick the in vivo tissue structure. To do this, we created nanopatterned PEG hydrogels and used these novel materials to direct cardiomyocyte organization in vitro. We then assessed the structural and functional characteristics of the resulting large-scale nanoengineered cardiac tissues. PEG nanostructured gels re-created 2-D cardiac tissues with an architecture that mimicked that of native tissue, key to many of its functional properties. Nanopatterned PEG hydrogels were fabricated using capillary force lithography. Precisely-defined nanopatterns of topological features and then ECM components were cast onto 22 mm diameter coverslips. Neonatal rat ventricular myocytes were seeded on these surfaces, forming confluent monolayers with controlled macroscopic alignment (anisotropy). The structural and functional properties of the nanoengineered cardiac tissue were then characterized. The nanoscale topographic features were found to regulate cell and cytoskeleton alignment and striation. In addition, immunocytochemical and ultrastructural analyses demonstrated that nanoscale control of cell-ECM interaction induces extensive geometrical alteration in the focal adhesions and gap junctions (connexin-43) formed in our engineered tissue, compared with conventional tissue monolayers of randomly distributed cardiac cells. Optical mapping of the integrative functional behavior of cultured monolayers revealed that monolayers formed on nanostructured substrata exhibited directional differences in conduction velocity, with an anisotropy ratio of 2.6±0.4 as compared with 1.0±0.1 (n=6) on control surfaces. Conduction velocity (CV) in the longitudinal direction (LCV, 26±5 cm/sec) was significantly faster than CV in the transverse direction (TCV, 10±2 cm/sec) for tissues formed on the nanostructured substrata (n=4, p<0.01). There were no directional differences in CV for control surfaces (LCV= 19±7 cm/sec, TCV = 19±7 cm/sec, n=6). In conclusion, we report a simple method to produce large-scale nanoengineered cardiac tissue components and illustrate the role of nanostructured ECM in defining the initial assembly of cardiac cells and the resulting tissue structure and function. Further electrophysiological studies of these nanoengineered cardiac tissues should provide novel insights into normal and diseased cardiac tissue function. In addition, the nanopatterning techniques employed here may be useful in creating tissues that mimic native cardiac architecture for therapeutic applications.