This paper presents a novel method for preparing nearly pure cultures of astrocytes and oligodendrocytes from rat cerebral tissue. The method involves the stratification of astrocytes and oligodendrocytes in culture, followed by selective detachment of oligodendrocytes through shaking cultures on an orbital shaker. The purity of the cultures is assessed using electron microscopy, enzyme assays, and pharmacological responsiveness. The results show that the cultures can be maintained for several weeks and are suitable for studying the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells. The method allows for the preparation of cultures with >98% purity and contains ~1–2 × 10^7 viable cells. The study also examines the influence of brain extract and dibutyryl cAMP on the morphology and ultrastructure of the cultures, finding that these agents induce astroglial process formation without affecting oligodendrocytes. The findings suggest that the method can effectively separate and maintain pure cultures of astrocytes and oligodendrocytes, facilitating further research in these areas.This paper presents a novel method for preparing nearly pure cultures of astrocytes and oligodendrocytes from rat cerebral tissue. The method involves the stratification of astrocytes and oligodendrocytes in culture, followed by selective detachment of oligodendrocytes through shaking cultures on an orbital shaker. The purity of the cultures is assessed using electron microscopy, enzyme assays, and pharmacological responsiveness. The results show that the cultures can be maintained for several weeks and are suitable for studying the biochemistry, physiology, and pharmacology of these two major classes of central nervous system cells. The method allows for the preparation of cultures with >98% purity and contains ~1–2 × 10^7 viable cells. The study also examines the influence of brain extract and dibutyryl cAMP on the morphology and ultrastructure of the cultures, finding that these agents induce astroglial process formation without affecting oligodendrocytes. The findings suggest that the method can effectively separate and maintain pure cultures of astrocytes and oligodendrocytes, facilitating further research in these areas.