The ultrastructure of astrocytes in an organotypic slice culture of the rat visual cortex was investigated using ultrathin sections and freeze-fracture replicas. After a culture period of 9-15 days, a glial scaffold formed that separated the bulk of the slice neuropil from the medium and the underlying plasma clot. However, the glial cells and processes did not build a dense barrier but allowed the outgrowth of neurites. A basal lamina covering the medium-oriented surface of the astrocytes was not found. In freeze-fracture replicas, orthogonal arrays of particles (OAP) were characteristic components of astrocytic membranes. The OAP density in membranes bordering the medium was 35 +/- 13 OAP/microns 2, corresponding to 2.5% of this membrane area; the OAP density in membranes within the slice neuropil was 22 +/- 12 OAP/microns 2, corresponding to 1.4% of this membrane area. Although the difference was significant, it was greatly reduced when comparing OAP densities in endfoot and non-endfoot membranes in vivo. Another node of polarity was recognized in astrocytes of the organotypic slice culture. In membranes of astrocytes bordering upon the medium, the density of non-OAP intramembranous particles (IMP) was clearly higher (1130 +/- 136 IMP/microns 2) than in membranes of astrocytes in the center of the slice (700 +/- 172 IMP/microns 2). This pronounced IMP-related polarity was observed neither in vivo nor in cultured astrocytes. The present study suggests, together with data from the literature, that the distribution of astrocytic OAP across the cell surface is influenced by the existence of a basal lamina and neuronal activity, and that astrocytes possess a more remarkable plasticity of membrane structure than previously suspected.