Brevican is one of the most abundant chondroitin sulfate proteoglycans in the adult rat brain. We have recently shown that the C-type lectin domain of brevican binds fibronectin type III domains 3-5 of tenascin-R. Here we report strong evidence for a physiological basis for this interaction. Substantial brevican immunoreactivity was detected in a number of nuclei and in the reticular formations throughout the midbrain and hindbrain, including, but not limited to, the deep cerebellar nuclei, the trapezoid body, the red nucleus, the oculomotor nucleus, the vestibular nucleus, the cochlear nucleus, the gigantocellular reticular nucleus, the motor trigeminal nucleus, and the lateral superior olive. Most of the brevican immunoreactivity exhibited pericellular and reticular staining patterns. In almost all of these sites, brevican immunoreactivity colocalized with that of tenascin-R, which was also substantially codistributed with versican, another member of the lectican family. Detailed analysis revealed that the pericellular staining of brevican resembled that in perineuronal nets in which tenascin-R has been localized. Immunoelectron microscopy identified brevican immunoreactivity in the intercellular spaces surrounding presynaptic boutons and on their surfaces, but not in the synaptic clefts or in their immediate vicinity, a distribution pattern consistent with perineuronal nets. Taken together, our results provide strong evidence that the previously reported interactions between brevican and tenascin-R may play a functional role within the perineuronal nets.