Alpha-adrenergic (αAR) stimulation induces hypertrophy in adult rat ventricular cardiomyocytes (ARVM) via a reactive oxygen species (ROS)-dependent mechanism. However, the precise species, source and/or site of effect of the ROS involved are not known. ARVM were infected with adenoviral vectors to overexpress superoxide dismutase (SOD) or catalase (Cat) in the cytosolic (SOD1, Cat) or mitochondrial (SOD2, msp-Cat) compartment. Hypertrophy was assessed by 3H-leucine incorporation 48 hours after αAR stimulation and the ratio of reduced to oxidized glutathione (GSH/GSSG) was determined as a measure of net oxidative stress. αAR stimulation for 24 hrs decreased the GSH/GSSG ratio from 37.7±6.2 to 18.0±3.7 (p<0.01, n = 4) indicative of increased oxidative stress. Catalase activity was increased from 0.11±0.02 mmol/mg-min in control cells to 0.43±0.13 mmol/mg-min (p<0.03 vs. control) in cells overexpressing msp-Cat and 1.71±0.38 (p<0.003 vs. control) in cells overexpressing Cat (p<0.02 vs. msp-Cat, n = 4). αAR-stimulated 3H-leucine incorporation (48 hrs, n = 7) was decreased from +62±5% in cells overexpressing lac-Z to +25±9% (p<0.005 vs. lac-Z) in cells overexpressing msp-Cat, but was unchanged in cells overexpressing Cat (+57±8%) or SOD1 (+51±7%). In contrast, overexpression of SOD2 increased αAR-stimulated 3H-leucine incorporation to +84±8% (p<0.05 vs. lac-Z). These data suggest that H2O2 or a derivative, rather than superoxide anion, is the primary mediator of ROS-dependent αAR-stimulated protein synthesis. Furthermore, the selective efficacy of mitochondrially-directed Cat, despite a lower overall catalase activity, suggests that αAR-stimulated protein synthesis is mediated by ROS made or acting in or near the mitochondria. Alpha-adrenergic (αAR) stimulation induces hypertrophy in adult rat ventricular cardiomyocytes (ARVM) via a reactive oxygen species (ROS)-dependent mechanism. However, the precise species, source and/or site of effect of the ROS involved are not known. ARVM were infected with adenoviral vectors to overexpress superoxide dismutase (SOD) or catalase (Cat) in the cytosolic (SOD1, Cat) or mitochondrial (SOD2, msp-Cat) compartment. Hypertrophy was assessed by 3H-leucine incorporation 48 hours after αAR stimulation and the ratio of reduced to oxidized glutathione (GSH/GSSG) was determined as a measure of net oxidative stress. αAR stimulation for 24 hrs decreased the GSH/GSSG ratio from 37.7±6.2 to 18.0±3.7 (p<0.01, n = 4) indicative of increased oxidative stress. Catalase activity was increased from 0.11±0.02 mmol/mg-min in control cells to 0.43±0.13 mmol/mg-min (p<0.03 vs. control) in cells overexpressing msp-Cat and 1.71±0.38 (p<0.003 vs. control) in cells overexpressing Cat (p<0.02 vs. msp-Cat, n = 4). αAR-stimulated 3H-leucine incorporation (48 hrs, n = 7) was decreased from +62±5% in cells overexpressing lac-Z to +25±9% (p<0.005 vs. lac-Z) in cells overexpressing msp-Cat, but was unchanged in cells overexpressing Cat (+57±8%) or SOD1 (+51±7%). In contrast, overexpression of SOD2 increased αAR-stimulated 3H-leucine incorporation to +84±8% (p<0.05 vs. lac-Z). These data suggest that H2O2 or a derivative, rather than superoxide anion, is the primary mediator of ROS-dependent αAR-stimulated protein synthesis. Furthermore, the selective efficacy of mitochondrially-directed Cat, despite a lower overall catalase activity, suggests that αAR-stimulated protein synthesis is mediated by ROS made or acting in or near the mitochondria.