Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca]) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca] and contractility. We tested the hypothesis that in the aged heart, [Ca] handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20⁻24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca], isolated cardiac myocytes were field-stimulated and [Ca] was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts ( < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca] in aged myocytes ( < 0.05). Time-50 [Ca] decay was increased in aged myocytes ( < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca sensitivity was reduced in aged myocytes ( < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment ( < 0.05). In conclusion, β-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca handling proteins and myofilaments.