One potential cell source for cardiac tissue engineering is embryonic stem cell-derived cardiomyocytes (ESC-CMs). However, obtaining pure cultures of cardiomyocytes in sufficient numbers to form large-scale tissues from these cells is challenging. In addition, the electrophysiological properties of tissues formed using ESC-CMs are unknown. This study used genetic selection to obtain pure cardiomyocyte cultures from mouse ESCs and examined the large-scale electrophysiological properties of these mESC-CMs monolayers (2 cm diameter). Methods: In order to obtain sufficient numbers of pure cardiomyocytes, mESCs were stably transfected with a DNA construct allowing expression of the neomycin resistance gene under the control of an -myosin heavy chain promoter (α-MHC, Klug et al., 1996). The mESCs were differentiated through embryoid body formation. Following neomycin selection, cells were dissociated on day 10 post-initiation of differentiation and seeded at confluency to form functional mESC-CM monolayers. On day 16-18 the monolayers were stained with voltage-sensitive dye di-4-ANEPPS and optically mapped at 253 sites in Tyrode's solution (36°C). Results: Differentiation and selection resulted in mESC-CMs that immunostained positively for the cardiac markers α-sarcomeric actinin, α-MHC, and cardiac troponin I. Obtained mESC-CMs beat in culture up to 5 months. Using optical mapping, planar wave propagation was observed across the confluent monolayers of mESC-CMs (n = 6). Planar conduction velocity was slow (2.4±0.9 cm/sec) compared to control neonatal rat cardiomyocytes (16±3 cm/sec), suggesting additional cues will be needed to further direct ESC-CMs toward the adult cardiomyocyte phenotype. This study demonstrates that cardiomyocytes derived from mESCs can be used to create electrophysiologically functional cardiac tissues. After additional electrophysiological characterization and optimization of cardiac phenotype, forming tissues from ESC-CMs may be a viable option for cardiac tissue engineering applications.