High titer polyclonal antibodies were produced in rabbit against a peptide unique to NaV1.2 sodium channels. NaV1.2 antibodies displayed 500,000-fold greater affinity for the NaV1.2 peptide compared with NaV1.1 or NaV1.3 peptides from the same region. These antibodies, when coupled to Sepharose beads, retained saxitoxin binding sites from solubilized rat brain membranes. Eluted protein from this antibody-affinity column was recognized by antibodies directed against neuronal voltage-gated sodium channels. Rabbit antibodies, which had been partially purified, were used in immunocytochemical localization of the NaV1.2 channel in 50 microm rat brain slices at dilutions of 1:1000 or 1:2000. NaV1.2 channels were predominately localized in unmyelinated fibers in the cortex, hippocampus, spinal cord and hypothalamus. Varicosities were seen in fiber staining which may reflect true varicosities in the fiber or simply varying densities of sodium channels along the fiber. Cell body staining with the NaV1.2 antibody was primarily observed in the hypothalamus. Antibody staining in the cerebellum was complex, with staining observed primarily in posterior lobes and considerably lower amounts of staining observed in anterior lobes. Specific staining was limited to fibers located in the granule and molecular layer, in an orientation consistent with granule cell unmyelinated axon labeling.