—The stimulatory GTP-binding protein Gsα transmits signals from catecholamine receptors to activate adenylyl cyclase and thereby initiate a cascade leading to cardiac chronotropy and inotropy. Transgenic mice overexpressing the Gs α subunit (Gsα) selectively in their hearts exhibit increased cardiac contractility in response to β-adrenergic receptor stimulation. However, with aging, these mice develop a cardiomyopathy. This study sought morphological and biochemical evidence that overexpression of Gsα is associated with increased myocyte apoptosis in the older animals and to determine whether such overexpression can promote apoptosis of isolated neonatal cardiac myocytes exposed to β-adrenergic receptor agonists. In the hearts of 15- to 18-month-old Gsα transgenic mice, histochemistry and electron microscopy illustrated the existence of numerous myocytes with abnormal nuclei embedded in collagen-rich connective tissue. Terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL, for in situ labeling of DNA breaks) demonstrated that ≈0.6% of myocyte nuclei contained fragmented DNA. Agarose gel electrophoresis provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation. Cultured cardiac myocytes from newborn Gsα transgenic mice showed increased TUNEL staining and internucleosomal DNA fragmentation compared with wild-type controls when treated with the β-agonist isoproterenol. Thus, enhanced activation of β-adrenergic signaling by overexpression of Gsα in the hearts of transgenic mice induces apoptosis of cardiac myocytes. This represents a potential mechanism that may contribute to the development of cardiomyopathy in this model.