In skeletal muscle, neuronal nitric oxide synthase is localized at the sarcolemma in association with the dystrophin glycoprotein complex (DGC). The nNOS N-terminal 231 amino acids comprise a PDZ domain (residues 1–100) and a β-hairpin finger loop (residues 101–130) which binds α-syntrophin located in the DGC. Endogenous nNOS and GFP-tagged nNOS localize to the sarcolemma in mouse C2C12 myotubes. Expression of GFP-tagged nNOS domains in C2C12 myotubes reveals that the PDZ domain and the β-hairpin finger loop of nNOS are independently capable of localizing to the sarcolemma of C2C12 myotubes. Binding studies indicate that α-syntrophin binds only to the β-hairpin finger loop and not the PDZ domain of nNOS. nNOS may bind to proteins in addition to α-syntrophin at muscle sarcolemma.