In early-stage heart, the cardiac impulse does not propagate through the specialized conduction system but spreads from myocyte to myocyte. We hypothesized that the gap junction protein connexin45 (Cx45) regulates early-stage contractions, because it is the only gap junction protein described in early hearts. Cx45-deficient (Cx45−/−) mice die of heart failure, concomitantly displaying other complex defects in the cardiovascular system. In order to determine the specific cardiac muscular function of Cx45, we created Cx45−/− embryonic stem (ES) cells to be differentiated into cardiac muscle in vitro. Unlike the coordinated contractions of wild-type cells, differentiated Cx45−/− cardiac myocytes showed high and irregular pulsation rates. Alterations of the electrophysiological properties of the Cx45−/− cardiac myocytes were indicated both by extracellular recording on planar multielectrode array probes and by intracellular Ca2+ recording of the fluorescent Ca2+ indicator fura-2. The in vitro system minimizes an influence of hemodynamic factors that complicate the phenotypes of Cx45−/− mice. Our results indicate that Cx45 is an essential connexin for coordinated conduction through early cardiac myocytes. © 2004 Wiley-Liss, Inc.