We recently reported that α₁-adrenoceptor (α₁-AR) stimulation induces hypertrophy via activation of the mitogen/extracellular signal-regulated kinase (MEK) 1/2-extracellular signal-regulated kinase (ERK) 1/2 pathway and generates reactive oxygen species (ROS) in adult rat ventricular myocytes (ARVM). Here we investigate the intracellular source of ROS in ARVM and the mechanism by which ROS activate hypertrophic signaling after α₁-AR stimulation. Pretreatment of ARVM with the ROS scavenger Mn(III)terakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP) completely inhibited the α₁-AR-stimulated activation of Ras-MEK1/2-ERK1/2. Direct addition of H₂O₂ or the superoxide generator menadione activated ERK1/2, which is also prevented by MnTMPyP pretreatment. We found that ARVM express gp91^phox, p22^phox, p67^phox, and p47^phox, four major components of NAD(P)H oxidase, and that α₁-AR-stimulated ERK1/2 activation was blocked by four structurally unrelated inhibitors of NAD(P)H oxidase [diphenyleneiodonium, phenylarsine oxide, 4-(2-aminoethyl)benzenesulfonyl fluoride, and cadmium]. Conversely, inhibitors for other potential ROS-producing systems, including mitochondrial electron transport chain, nitric oxide synthase, xanthine oxidase, and cyclooxygenase, had no effect on α₁-AR-stimulated ERK1/2 activation. Taken together, our results show that ventricular myocytes express components of an NAD(P)H oxidase that appear to be involved in α₁-AR-stimulated hypertrophic signaling via ROS-mediated activation of Ras-MEK1/2-ERK1/2.