The addition of glutamine as a major nutrient to cultured neonatal rat cardiomyocytes produced an increase in myocyte size and the organization of actin into myofibrillar arrays. The cellular response was associated with increased abundance of the mRNAs encoding the contractile proteins, alpha-myosin heavy chain and cardiac alpha-actin, and the metabolic enzymes, muscle carnitine palmitoyl transferase I and muscle adenylosuccinate synthetase (ADSS1). Adss1 gene expression was induced approximately 5-fold in glutamine-treated rat neonatal cardiac myocytes. The induction was mediated through the protein kinase A and mammalian target of rapamycin signaling pathways and required a cyclic AMP response element associated with the promoter region of the Adss1 gene. These results highlight glutamine as a major nutrient regulator of cardiac gene expression and identify protein kinase A and mammalian target of rapamycin signaling pathways as mediators of the cardiomyocyte transcriptional response.