Recent evidence suggests that increased superoxide production may decrease the bioavailability of endothelial nitric oxide synthase (eNOS)-derived nitric oxide in the myocardium of neuronal nitric oxide synthase (nNOS)^−/− mice. To test the functional relevance of this observation, we compared the eNOS-dependent inotropic and [Ca²⁺]_i transient effect of beta3-adrenergic receptor (β3-AR) stimulation in left ventricular myocytes from eNOS^−/− and nNOS^−/− mice and their wild-type littermates. β3-AR stimulation (with BRL 37344, 10 μmol/l in the presence of the β1 and β2-AR blocker nadolol, 10 μmol/l) resulted in a significant reduction in cell shortening and [Ca²⁺]_i transient amplitude in wild-type myocytes. In contrast, BRL plusnadolol had no effect in myocytes from eNOS^−/− and nNOS^−/− myocytes (in the absence of changes in β3-AR messenger RNA levels or in eNOS protein expression in the latter) or in nNOS^+/+ myocytes pretreated with the nNOS-specific inhibitor, SMTC (0.1 μmol/l). Inhibition of xanthine oxidoreductase (XOR) or NADPH oxidases caused a similar reduction in basal superoxide production in nNOS^−/− myocytes; however, whereas apocynin had no effect, XOR inhibition restored the negative inotropic response to β3-AR stimulation in nNOS^−/− myocytes but not in eNOS^−/− myocytes. eNOS activity was uncoupled in the myocardium of nNOS^−/− mice in the absence of changes in BH4 and biopterin levels or arginase activity. However, l-arginine transport was impaired in nNOS^−/− myocytes and excess l-arginine (10 mmol/l) restored the response to β3-AR stimulation. In summary, increased superoxide production by XOR (but not by NADPH oxidase) selectively abolished the negative inotropic effect of β3-AR stimulation in nNOS^−/− myocytes by decreasing l-arginine transport and the bioavailability of eNOS-derived nitric oxide. These findings demonstrate that the source and subcellular localisation ofsuperoxide production can account for the diverse and specialised actions of reactive oxygen species in the heart.