Muscular dystrophies that arise from mutations of genes that encode proteins in the dystrophin–glycoprotein complex (DGC) frequently involve defects in the structure of neuromuscular junctions (NMJs). DGC mutations that cause NMJ defects typically cause a secondary loss of neuronal nitric oxide synthase (nNOS) from the post-synaptic membrane. We tested the hypothesis that reduction of muscle-derived NO production causes NMJ defects in DGC mutants by analyzing the effect of modulating muscle NO production on NMJ structure in mutant and wild-type muscles. We found that nNOS null mutants, dystrophin-deficient mdx mice and α-syntrophin null mutants showed reductions in the concentration of acetylcholine receptors (AChRs) at the post-synaptic membrane. Also, expression of a muscle-specific NOS transgene increased AChR concentration, which reflected an increase in both AChR expression and clustering. NOS transgene expression also increased the size of NMJs, and partially corrected defects in normal NMJ architecture that were observed in mdx and α-syntrophin null muscles. In addition, stimulation of AChR clustering in vitro by application of laminin or VVA B4 lectin induced a 3–4-fold increase in NOS activity and increased AChR clustering that could be prevented by NOS inhibition. However, the partial rescue of NMJ structure by expression of a NOS transgene required the expression of α- or β1-syntrophin at the NMJ; partial NMJ rescue was seen in the muscles of α-syntrophin mutants that expressed β1-syntrophin, but no rescue was observed in muscles of α-syntrophin mutants that also lacked β1-syntrophin. These findings show that NO promotes AChR expression and clustering in vivo and contributes to normal NMJ architecture. The results suggest that defects in NMJ structure that occur in some DGC mutants can result from the secondary loss of NOS from muscle.