Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have the potential to supply large numbers of cells for cardiac regeneration. In this study, we characterize for the first time the electrophysiologial properties of tissues formed from ESC-CMs as the percentage of ESC-CMs increases from 10% to 100%. Methods: A pure population of ESC-CMs was obtained by stably transfecting mouse ESCs with a DNA construct expressing the neomycin resistance gene under the control of an α-myosin heavy chain promoter (Klug et al., 1996). Following neomycin selection, ESC-CMS were dissociated on day 10 of differentiation and seeded as large-area (3.8 cm²) confluent monolayers either at a 0%, 10% or 30% mix with neonatal rat ventricular myocytes(NRVMs) or as a pure population (100%). 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: All tissue sheets exhibited synchronous, spontaneous contractions in culture for at least 1 week and in some cases, up to 5 months. Both co-culture and pure ESC-CM monolayers could be electrically paced with maximum capture rates of up to 6 Hz. For co-culture monolayers, action potential duration (APD) was relatively independent of the percentage of ESC-CMs incorporated (0%: 130±30 ms, n=3; 10%: 158±60 ms, n=4; 30%: 156±17 ms, n=4, at 2 Hz pacing, p>0.2 between groups). However, conduction velocity (CV) decreased with increasing percentage of ESC-CMs incorporated (0%: 19±7 cm/s, n=3; 10%: 11±3 cm/s, n=4; 30%: 8±1 cm/s, n=4, at 2 Hz). Pure ESC-CM monolayers immunostained positively for connexin 43 and for cardiac markers including α-sarcomeric actinin, α-MHC, and cardiac troponin I. CV in pure ESC-CM monolayers was 3.8±1.4 cm/s(n=5) at 2 Hz pacing and did not change significantly with increasing pacing rate (4.0±1.4cm/s at 6 Hz,n=3). APD was 109±11 ms (n=5) at 2 Hz pacing and decreased with increasing pacing rate (90±5 ms at 6 Hz, n=3). Conclusion: Our study shows for the first time that ESC-CMs can electrically integrate with NRVMs over a large area and by themselves, can form an electrophysiologically functional tissue substrate. These results support the idea that ESC-CMs may be a viable cell source for cardiac regeneration.