Studies have shown that neuronal nitric oxide synthase (nNOS, NOS1) knockout mice (NOS1^-/-)have increased or decreased contractility, but consistently have found a slowed rate of [Ca²⁺]_i decline and relaxation. Contraction and [Ca²⁺]_i decline are determined by many factors, one of which is phospholamban (PLB). The purpose of this study is to determine the involvement of PLB in the NOS1-mediated effects. Force-frequency experiments were performed in trabeculae isolated from NOS1^-/- and wildtype (WT) mice. We also simultaneously measured Ca²⁺ transients (Fluo-4) and cell shortening (edge detection) in myocytes isolated from WT, NOS1^-/-, and PLB^-/- mice. NOS1^-/- trabeculae had a blunted force-frequency response and prolonged relaxation. We observed similar effects in myocytes with NOS1 knockout or specific NOS1 inhibition with S-Methyl-L-thiocitrulline (SMLT) in WT myocytes (i.e., decreased Ca²⁺ transient and cell shortening amplitudes and prolonged decline of [Ca²⁺]_i). Alternatively, NOS1 inhibition with SMLT in PLB^-/- myocytes had no effect. Acute inhibition of NOS1 with SMLT in WT myocytes also decreased basal PLB Serine16 phosphorylation . Furthermore, there was a decreased SR Ca²⁺ load with NOS1 knockout or inhibition, which is consistent with the negative contractile effects. Perfusion with FeTPPS (peroxynitrite decomposition catalyst) mimicked the effects of NOS1 knockout or inhibition. -adrenergic stimulation restored the slowed [Ca²⁺]_i decline in NOS1^-/- myocytes, but a blunted contraction remained, suggesting additional protein target(s). In summary, NOS1 inhibition or knockout leads to decreased contraction and slowed [Ca²⁺]_i decline, and this effect is absent in PLB^-/- myocytes. Thus, NOS1 signaling modulates PLB Serine16 phosphorylation, in part, via peroxynitrite.