Astrocytes are the main glial cells maintaining water-electrolyte and energy balance in the brain. Today, astroglia is also believed to take a direct part in the regulation of synaptic transmission and in enabling synchronous operation of neurons at large distances. Astrocytes fulfil their functions through numerous processes that penetrate the entire neuropil. The authors believe that changes in the astrocyte membrane surface area per unit volume of neuropil directly reflect changes in the intensity of the astrocyte–neuron interaction. Strengthening or weakening of astrocyte regulation, undoubtedly, affect the functioning of neural circuits. Nevertheless, in spite of the growing popularity of research into the glia–neuron relations, this aspect remains insufficiently studied when it comes to the cerebral cortex. The purpose of this study was to layer-by-layer determine the astrocyte membrane surface per unit volume in the neuropil of the rat primary somatosensory cortex. The research was conducted on samples of the primary somatosensory cortex obtained from 5 white male rats (P60–80). After immune labeling against astrocytic marker S100B using the pre-embedding method, the samples were prepared for transmission electron microscopy according to the standard technique. In total, 250 electron micrographs were obtained for each layer of the primary somatosensory cortex, which were then used to determine the astrocyte membrane surface area per unit volume in the neuropil by means of the random secant method. The research found that this indicator is the minimum in the first and maximum in the fifth layers of the cortical column. In addition, the article discusses the possible functional consequences of uneven distribution of astrocytic membranes in the neocortex.