Objective: Intercellular coupling in cardiac myocytes is mediated via gap junctions. Gap junctions are essential to the coordinated contraction of the heart, and decreased coupling can lead to arrhythmias. The gap junction protein connexin 43 (Cx43) is degraded via both the proteasomes and lysosomes, with ubiquitination being a trigger of endocytosis. Stimulation of Gaq receptors in cardiomyocytes decreases coupling by a decrease in phosphatidylinositol 4,5 bisphosphat in the plasma membrane. The objective of this study was to investigate the role of the proteasomes and lysosomes in the internalization and degradation of Cx43 in rat cardiomyocytes after Gaq stimulation. Methods: Coupling was measured by dye transfer of Lucifer yellow after electroporation into cultured neonatal rat cardiomyocytes. Junctional and non-junctional Cx43 was separated by Triton X-100 fractionation. Ubiquitination of Cx43 was investigated by immunoprecipitation and western blot. Results: Stimulation of cardiomyocytes with the Gaq agonist norepinephrine (NE) for 10 min reduced coupling to 72% compared to control cells, whereas pre-treatment with the proteasomal inhibitor MG132 or the lysosomal inhibitor leupeptin restored coupling. Western blots of total, Triton X-100 soluble, and insoluble fractions of Cx43 showed that NE increased the soluble fraction, which is considered a measure of non-junctional Cx43. Unexpectedly, pre-treatment with MG132 and leupeptin increased this fraction further. NE also increased ubiquitination of Cx43. Conclusions: The proteasomal inhibitor MG132 has been shown to prevent internalization of Cx43. The dye transfer studies thus suggest that the proteasomes and lysosomes are involved in NE mediated internalization and degradation of Cx43. The increased ubiquitination is consistent with the internalization hypothesis. However, MG132 and leupeptin did not prevent the increase in the Triton X-100 soluble fraction of Cx43 induced by NE, suggesting that this fraction is not a definite measure of the non-junctional pool of Cx43.