Structural and functional cardiac anisotropy varies with the development, location, and pathophysiology in the heart. The goal of this study was to design a cell culture model system in which the degree, change in fiber direction, and discontinuity of anisotropy can be controlled over centimeter-size length scales. Neonatal rat ventricular myocytes were cultured on fibronectin on 20-mm diameter circular cover slips. Structure-function relationships were assessed using immunostaining and optical mapping. Cell culture on microabraded cover slips yielded cell elongation and coalignment in the direction of abrasion, and uniform, macroscopically continuous, elliptical propagation with point stimulation. Coarser microabrasion (wider and deeper abrasion grooves) increased longitudinal (23.5 to 37.2 cm/s; r=0.66) and decreased transverse conduction velocity (18.1 to 9.2 cm/s; r=-0.84), which resulted in increased longitudinal-to-transverse velocity anisotropy ratios (1.3 to 3.7, n=61). A thin transition zone between adjacent uniformly anisotropic areas with 45 degrees or 90 degrees difference in fiber orientation acted as a secondary source during 2x threshold field stimulus. Cell culture on cover slips micropatterned with 12- or 25- micro m wide fibronectin lines and previously coated with decreasing concentrations of background fibronectin yielded transition from continuous to discontinuous anisotropic architecture with longitudinally oriented intercellular clefts, decreased transverse velocity (16.9 to 2.6 cm/s; r=-0.95), increased velocity anisotropy ratios (1.6 to 5.6, n=70), and decreased longitudinal velocity (36.4 to 14.6 cm/s; r=-0.85) for anisotropy ratios >3.5. Cultures of cardiac myocytes with controlled degree, uniformity and continuity of structural, and functional anisotropy may enable systematic 2-dimensional in vitro studies of macroscopic structure-related mechanisms of reentrant arrhythmias. The full text of this article is available at http://www.circresaha.org.