Background — The major source of nitric oxide (NO) in the heart is the constitutive form of NO synthases (eNOS, NOS III) that is expressed in vascular endothelium and cardiac myocytes. NO mediates endothelium-dependent vasodilation and may modulate cardiac function. We examined the role of NO in hearts from transgenic (TG) mice overexpressing eNOS exclusively in cardiac myocytes. Methods and Results — Three independent TG lines with varying levels of NOS activity were selected, and the hearts were isolated and retrogradely perfused at constant flow. We found that NO is positively inotropic in spontaneously beating hearts from wild-type (WT) mice, whereas hearts overexpressing eNOS had reduced basal contractility that was partially reversed by NOS blockade. Heart rate was not altered. Acetylcholine (10 to 1000 nmol/L) increased contractility in unstimulated hearts and decreased contractility after β-adrenergic stimulation with norepinephrine, and these responses were identical in WT and TG hearts. Finally, resting systolic intracellular calcium (Ca ²⁺ _i ) tended to be lower in TG than in WT hearts, and the beat-to-beat responsiveness to Ca ²⁺ _i was reduced in hearts with eNOS overexpression. Conclusions — High levels of endogenous myocyte-derived NO blunt myofilament Ca ²⁺ sensitivity. The similar effects of acetylcholine on contractility and heart rate, as well as the identical basal intrinsic heart rate in WT and TG hearts, provide a solid argument against NO as an important modulator of neurohormonal control of myocardial function.