The neuromuscular junction (NMJ) is a regionally specialized area of myofibers defined, in part, by specific gene expression from underlying myonuclei. We sought to obtain a more complete picture of the mRNA transcripts and proteins playing a role in NMJ formation and maintenance using laser capture microdissection (LCM) and to define expression profiles of the nuclear domain at the NMJ. NMJs (800) were isolated from normal mouse tibialis anterior muscle by LCM, with an equal amount of adjacent non-NMJ regions isolated. Many known components of the NMJ were found significantly differentially expressed. Three differentially expressed potential novel components of the NMJ were chosen for further study, and each was validated by immunostaining with and without blocking peptides (3/3), quantitative RT-PCR (3/3), and in situ hybridization (1/3). The three genes validated were dual-specificity phosphatase-6 (DUSP6), ribosomal receptor-binding protein-1 (RRBP1), and vacuolar protein sorting-26 (VPS26). Query of each of these novel components in a 27-time point in vivo muscle regeneration series showed expression commensurate with previously known NMJ markers (nestin, alpha-ACh receptor). Understanding and discovering elements responsible for the integrity and function of NMJs is relevant to understanding neuromuscular diseases such as spinal muscular atrophy. Our LCM-based mRNA expression profiling provided us with new means of identification of specific genes potentially responsible for NMJ stability and function and new candidates for involvement in disease pathogenesis.