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 Ca(2+) recording of the fluorescent Ca(2+) 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. The Supplementary Material referred to in this article can be found at the Anatomical Record website (http://www.interscience.wiley.com/jpages/0003-276X/suppmat).