The mechanisms by which nitric oxide (NO) influences myocardial Ca²⁺ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca²⁺ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dt_max normalized to end-diastolic volume; dP/dt-EDV) by 51±5% (P<0.001) and lusitropy (tau; τ) by 20.3±2.0% (P<0.05). These responses were markedly attenuated in mice lacking NOS1 (NOS1^−/−) (15±2% increase in dP/dt-EDV; P<0.001 versus WT; and no change in τ; P<0.01 versus WT). Isolated myocytes from NOS1^−/− (≈2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca²⁺ transients ([Ca²⁺]_i) compared with WT. SR Ca²⁺ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca²⁺]_i at 4 Hz increased 107±23% above 1 Hz response) but not in NOS1^−/− (13±26%; P<0.01 versus WT). In contrast, mice lacking NOS3 (NOS3^−/−) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca²⁺]_i, and SR Ca²⁺ stores. NOS1^−/− had increased SR Ca²⁺ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca²⁺ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.