Nitric oxide (NO) functions principally as a diffusible paracrine effector. The exception is in cardiomyocytes where both NO synthases (NOS) and target proteins coexist, allowing NO to work in an autocrine/intracrine fashion. However, the most abundant myocyte isoform (NOS3) is far more expressed in vascular endothelium; thus, the in vivo contribution of myocyte-NOS3 remains less clear. The present study tested this role by transfecting whole hearts of NOS3-null (NOS3^−/−) mice with adenovirus-expressing NOS3 coupled to a α-MHC promoter (AdV_NOS3), comparing results to hearts transfected with marker-gene β-galactosidase (AdVβ_gal). Total myocardial NOS3 protein and activity were restored to near wild-type (WT) levels in NOS3^−/−+AdV_NOS3 hearts, and NOS3 relocalized normally with caveolin-3. Ejection function by pressure-volume analysis was enhanced in NOS3^−/−+AdVβ_gal over WT or NOS3^−/−+AdV_NOS3. More prominently, isoproterenol (ISO)-stimulated systolic and diastolic function in WT was amplified in NOS3^−/−+AdVβ_gal, whereas NOS3^−/−+AdV_NOS3 returned the response to control. ISO-activated systolic function was inhibited 85% by concomitant muscarinic stimulation (carbachol) in NOS3^−/−+AdV_NOS3 but not NOS3^−/−+AdVβ_gal hearts. Lastly, NOS3^−/−+AdVβ_gal mice displayed enhanced inotropy and lusitropy over WT at slower heart rates but a blunted rate augmentation versus controls. A more positive rate response was restored in NOS3^−/−+AdV_NOS3 (P<0.001). Thus, myocyte autocrine/intracrine NOS3 regulation in vivo can underlie key roles in β-adrenergic, muscarinic, and frequency-dependent cardiac regulation.