Astrocytes are the major population of glial cells, and are key players in the development, maintenance, and functioning of the central nervous system (CNS). Their potential as targets of therapeutic intervention following CNS injury makes the elucidation of their cellular and subcellular physiology a primary research goal. Well defined and pure cell culture systems are required to examine astrocytic physiology, biochemical pathways and underlying responses to pathophysiologically altered conditions. Previously published protocols for establishing primary astrocyte cultures are time- and resource-consuming or suffer high contamination from other undesired cell types. Here we describe a new and simple procedure for producing highly pure (>99%) rat primary astrocyte cultures. The method involves a simple mechanical dissociation of harvested spinal cord tissue through a porous membrane and the subsequent plating of the cells on plain, untreated glass coverslips. Astrocytes adhere very well to the untreated glass while other cell types require a substrate such as poly-l-lysine. The method described here is, therefore, ideal for experiments which require highly pure astrocyte cultures.