Objective: To compare gap junction expression and intercellular coupling in wildtype neonatal cardiac myocytes to those from mice lacking the most abundant cardiac gap junction protein (connexin43, Cx43). Methods: Northern and Western blots compared connexin mRNA and protein levels, immunocytochemistry evaluated connexin distribution in neonatal Cx43 null(−/−), heterozygous(+/−) and wildtype(+/+) mouse hearts. Ca2+ imaging, dye coupling and electrophysiological methods evaluated intercellular communication. Results: Similar levels of Cx40 and Cx45 were detected in all genotypes, although in adult cardiac tissue from wildtype mice, Cx43 expression was higher than in heterozygotes. After culturing dissociated cells for 3–4 days, cardiocytes beat spontaneously; in Cx43(+/+) and (+/−) cultures, the beating was generally quite synchronous. In Cx43(−/−) mice, interbeat intervals were on average twice as long and more variable than in Cx43(+/+) or Cx43(+/−) cultures. Junctional conductance was lower by about 60% in Cx43(−/−) as compared to Cx43(+/−) and (+/+) littermates; Lucifer Yellow dye coupling was virtually absent in Cx43(−/−) cardiomyocytes but was comparably strong in wildtype and heterozygous siblings. Macroscopic junctional conductance measurements on Cx43(−/−) cardiocytes showed slightly stronger voltage sensitivity in these cells than in Cx43(+/+) cardiocytes. Unitary junctional conductance measurements revealed distinct populations of channels contributing to macroscopic conductance for Cx43(+/+) and Cx43(−/−) genotypes. Conclusions: In Cx43-deficient cardiac myocytes, the expression of other connexins only partially compensates for the functional loss, with dye coupling and spontaneous beating being strongly impaired.