Studies of the structural-functional organization of the boundaries of CNS areas address a fundamental task in neurobiology. Given the contradictory data on the structure of the superficial layers of the mammalian neocortex, there is value in undertaking detailed investigations of the structures and cytochemical organization of astrocytes – the main components of the barrier system of the brain – in animals used in experimental studies modeling diseases and brain trauma. The aim of the present work was to study the structural organization of astrocytes in layer I of the rat neocortex. Astrocytes were detected immunocytochemically using antibodies to glial fibrillary acidic protein, vimentin, and nestin, using light and confocal laser microscopy. The results show that the superficial glial limiting membrane (SGLM) has significant structural differences in different areas of the cortex and that astrocytes in layer I of the rat cerebral cortex are not typical protoplasmic astrocytes characteristic of the gray matter. These regional features in the organization of the SGLM are probably due to differences in the functional characteristics of the CSF:brain barrier in different areas of the brain.