Reactive oxygen species (ROS) play a crucial role in various physiological and pathological processes mediated by (beta-adrenergic receptors (beta-ARs) in cardiomyocytes. However, the sources and signaling pathways involved in ROS production induced by acute (beta-AR activation have not yet been fully defined. In primary neonatal mouse cardiomyocytes (NMCM5), the (beta-AR agonist isoproterenol (ISO) induced a rapid increase in mitochondrial ROS and total ROS production. Both the expression and activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2/4 (NOX 2/4) remained unchanged after 2 h of ISO treatment, suggesting that acute ISO stimulation mainly induces mitochondrial ROS production in NMCM5. Knockdown of beta-arrestin1, but not beta-arresting, inhibited ISO-induced mitochondrial ROS production within 1-2 h after ISO treatment. Moreover, forskolin, an adenylyl cyclase (AC) activator, rapidly increased mitochondrial ROS as early as 15 min after ISO treatment. Inhibition of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway abolished the mitochondrial ROS production within 15-60 min after ISO treatment. In conclusion, mitochondria are the major source of ROS production upon acute ISO stimulation. (beta-arrestin1, but not (beta-arresting, is involved in ISO-induced mitochondrial ROS production. Upon acute (beta-AR stimulation in NMCMs, the classical cAMP/PKA pathway is responsible for faster mitochondrial ROS production, whereas beta-arrestinl signaling is responsible for slower mitochondrial ROS production. (C) 2017 Elsevier Inc. All rights reserved.