Recent studies using molecular and genetic techniques just have started to elucidate the complex process that drives successful peripheral nerve regeneration. Introducing proteomics to this field, we unilaterally performed a facial nerve axotomy in 13 adult Wistar rats. Seven days later, a total of 40 20-microm coronary cryostat sections of the operated and contralateral unoperated nucleus facialis were microdissected. On the one hand, microdissected areas were pooled for each side, lysed and applied to ProteinChip Arrays. On the other hand, one microdissected area from the right and left facial nucleus each was directly placed on the affinity chromatographic array. Facial motoneurons were lysed in situ and released their proteins to spatially defined points. 215 laser addressable distinct positions across the surface of the spot enabled a high spatial resolution of measured protein profiles for the analysed tissue area. Protein profiles of the single positions were plotted over the used tissue section to visualize their distribution. The comparative analysis of the protein lysates from operated and normal nuclei facialis revealed, for both approaches used, differentially expressed proteins. Although by direct application of one cryostat section only a few hundred motoneurons were analysed, results comparable to these using lysates were obtained. Additionally, the applied technique revealed differences in the intensity distribution of several proteins of unknown function in the lesioned in comparison to the contralateral normal facial nucleus. This proteomic analysis with ultra high sensitivity paired with potential for a spatial resolution is a promising methodology for peripheral nerve regeneration studies.