A novel method has been developed to prepare nearly pure cultures of astrocytes and oligodendrocytes from postnatal rat cerebral tissue. The method relies on the absence of viable neurons in cultures from postnatal rats, the stratification of astrocytes and oligodendrocytes in culture, and the selective detachment of oligodendrocytes using shaking on an orbital shaker for 15–18 hours at 37°C. Cultures are >98% pure and contain ~1–2×10⁷ viable cells. Characterization methods include electron microscopy, enzymatic markers (CNPase and GPDH), and pharmacological responses. These cultures can be maintained for several weeks and are useful for studying the biochemistry, physiology, and pharmacology of central nervous system cells. The method involves preparing mixed glial cultures from 1- to 2-day-old rat pups, allowing them to grow for 7–9 days, and then shaking the cultures to detach oligodendrocytes. The resulting cultures are then separated and characterized. Astrocytes and oligodendrocytes can be distinguished by their morphology, ultrastructure, and enzymatic markers. Astrocytes are larger, have fewer processes, and are more proliferative, while oligodendrocytes are smaller, have more processes, and are less proliferative. The method allows for the preparation of essentially pure cultures of both cell types, which can be used for biochemical and pharmacological studies. The method is effective in separating astrocytes and oligodendrocytes, as evidenced by their distinct morphological and biochemical characteristics. Astrocytes are identified by their large size, lack of processes, and high proliferation rate, while oligodendrocytes are smaller, have more processes, and are less proliferative. The method also allows for the study of the effects of brain extract and dibutyryl cAMP on the morphology and ultrastructure of these cells. These studies indicate that astrocytes can form processes in response to these agents, while oligodendrocytes remain unaffected. The results suggest that essentially pure cultures of astrocytes and oligodendrocytes can be prepared and maintained, which should significantly aid in the study of these two major classes of central nervous system cells.A novel method has been developed to prepare nearly pure cultures of astrocytes and oligodendrocytes from postnatal rat cerebral tissue. The method relies on the absence of viable neurons in cultures from postnatal rats, the stratification of astrocytes and oligodendrocytes in culture, and the selective detachment of oligodendrocytes using shaking on an orbital shaker for 15–18 hours at 37°C. Cultures are >98% pure and contain ~1–2×10⁷ viable cells. Characterization methods include electron microscopy, enzymatic markers (CNPase and GPDH), and pharmacological responses. These cultures can be maintained for several weeks and are useful for studying the biochemistry, physiology, and pharmacology of central nervous system cells. The method involves preparing mixed glial cultures from 1- to 2-day-old rat pups, allowing them to grow for 7–9 days, and then shaking the cultures to detach oligodendrocytes. The resulting cultures are then separated and characterized. Astrocytes and oligodendrocytes can be distinguished by their morphology, ultrastructure, and enzymatic markers. Astrocytes are larger, have fewer processes, and are more proliferative, while oligodendrocytes are smaller, have more processes, and are less proliferative. The method allows for the preparation of essentially pure cultures of both cell types, which can be used for biochemical and pharmacological studies. The method is effective in separating astrocytes and oligodendrocytes, as evidenced by their distinct morphological and biochemical characteristics. Astrocytes are identified by their large size, lack of processes, and high proliferation rate, while oligodendrocytes are smaller, have more processes, and are less proliferative. The method also allows for the study of the effects of brain extract and dibutyryl cAMP on the morphology and ultrastructure of these cells. These studies indicate that astrocytes can form processes in response to these agents, while oligodendrocytes remain unaffected. The results suggest that essentially pure cultures of astrocytes and oligodendrocytes can be prepared and maintained, which should significantly aid in the study of these two major classes of central nervous system cells.