The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions. J. Comp. Neurol. 441:302–323, 2001. © 2001 Wiley-Liss, Inc.