To screen for new region-specific protein markers we compared the proteome maps of the primary visual and somatosensory areas V1 and S1 in mouse brain using 2-D difference gel electrophoresis (2-D DIGE). Twenty-three protein spots showed a statistically significant difference in expression level between V1 and S1, with 52% appearing more abundantly in V1. Twenty-six proteins were mass spectrometrically identified in 22 spots. To assess the validity of this list of potential areal markers generated by 2-D DIGE, the effective area-specific distribution profile of creatine kinase brain subtype (CKB), a protein with a clearly higher expression level in S1, was monitored with in situ hybridization. The mRNA expression profile of CKB displayed a clear area-specific distribution, which allowed demarcation of S1 and its topographical borders with neighboring neocortical areas. This proteomic study demonstrates the innovative application of 2-D DIGE and MS to select new regional markers for neuroscience research.