We describe a method to prepare postnatal rat brain primary cell cultures composed of astrocytes, oligodendrocytes, and microglia. After 1 week in vitro, the mixed glial cell cultures are free of neurons, meningeal cells and fibroblasts. We developed a simple procedure to selectively harvest enriched populations of each of the three major glial cell types. Because these cells are at a progenitor/immature stage, each can be further cultured separately in serum or serum-free media to yield large quantities of the desired glial cell subpopulations with a high degree of purity in the range of 96-99%. These cell culture models have been used extensively for performing biochemical, molecular, and pharmacological studies using standard assays and obtain sound quantitative data. These studies have given us insights into the development, properties, and functions of rat and mouse glial cells in vitro. The findings have largely been validated and extended in animal models over the last 3 decades. Since this method has been cited in more than 2,500 research papers, the data obtained across laboratories can be compared more readily.