A neuronal isoform of nitric oxide synthase (nNOS) has recently been located to the cardiac sarcoplasmic reticulum (SR). Subcellular localization of a constitutive NOS in the proximity of an activating source of Ca²⁺ suggests that cardiac nNOS-derived NO may regulate contraction by exerting a highly specific and localized action on ion channels/transporters involved in Ca²⁺ cycling. To test this hypothesis, we have investigated myocardial Ca²⁺ handling and contractility in nNOS knockout mice (nNOS^−/−) and in control mice (C) after acute nNOS inhibition with 100 μmol/L L-VNIO. nNOS gene disruption or L-VNIO increased basal contraction both in left ventricular (LV) myocytes (steady-state cell shortening 10.3±0.6% in nNOS^−/− versus 8.1±0.5% in C; P<0.05) and in vivo (LV ejection fraction 53.5±2.7 in nNOS^−/− versus 44.9±1.5% in C; P<0.05). nNOS disruption increased I_Ca density (in pA/pF, at 0 mV, −11.4±0.5 in nNOS^−/− versus −9.1±0.5 in C; P<0.05) and prolonged the slow time constant of inactivation of I_Ca by 38% (P<0.05), leading to an increased Ca²⁺ influx and a greater SR load in nNOS^−/− myocytes (in pC/pF, 0.78±0.04 in nNOS^−/− versus 0.64±0.03 in C; P<0.05). Consistent with these data, [Ca²⁺]_i transient (indo-1) peak amplitude was greater in nNOS^−/− myocytes (410/495 ratio 0.34±0.01 in nNOS^−/− versus 0.31±0.01 in C; P<0.05). These findings have uncovered a novel mechanism by which intracellular Ca²⁺ is regulated in LV myocytes and indicate that nNOS is an important determinant of basal contractility in the mammalian myocardium. The full text of this article is available at http://www.circresaha.org.