Astrocytes are known to play an integral role in the development of compartmental boundaries in the brain and in the creation of trauma-induced boundaries. However, the physical relationship between astrocytes and such boundaries in the adult brain is less clear. If astrocytes do respect or play an ongoing role in maintaining such boundaries, a correlation between the position of such a boundary and the morphology of neighboring astrocytes might be observable. In this study, we examined the distribution of astrocytes with respect to the laminar boundaries compartmentalizing afferents to the dentate gyrus molecular layer. In addition, we attempted to determine whether astrocyte morphology is influenced by these laminar boundaries. To this end, protoplasmic astrocytes in the adult rat dentate gyrus were revealed with fluorescent tracer dyes and subsequently analyzed with respect to laminar boundaries demarcated by means of immunolabeling for the lamina-specific molecules EphA4 and neural cell adhesion molecule (N-CAM). We find that astrocyte distribution is influenced by the boundary separating the associational/commissural and perforant path afferents. In addition, we show that astrocytes in this region are polarized in their morphology, unlike typically stellate astrocytes, but that the laminar boundaries themselves do not appear to confer this morphology. This polarized morphology, however acquired, may have import for the functioning of astrocytes within the highly organized composition of the dentate gyrus molecular layer and for the overall microphysiology of this and other brain regions.