Astrocytes show large morphological and functional heterogeneity and are involved in many aspects of neural function. Progress in defining astrocyte subpopulations has been hampered by the lack of a suitable antibody for their direct detection and isolation. Here, we describe a new monoclonal antibody, ACSA‐1, which was generated by immunization of GLAST1 knockout mice. The antibody specifically detects an extracellular epitope of the astrocyte‐specific L‐glutamate/L‐aspartate transporter GLAST (EAAT1, Slc1a3). As shown by immunohistochemistry, immunocytochemistry, and flow cytometry, ACSA‐1 was cross‐reactive for mouse, human, and rat. It labeled virtually all astrocytes positive for GFAP, GS, BLBP, RC2, and Nestin, including protoplastic, fibrous, and reactive astrocytes as well as Bergmann glia, Müller glia, and radial glia. Oligodendrocytes, microglia, neurons, and neuronal progenitors were negative for ACSA‐1. Using an immunomagnetic approach, we established a method for the isolation of GLAST‐positive cells with high purity. Binding of the antibody to GLAST and subsequent sorting of GLAST‐positive cells neither interfered with cellular glutamate transport nor compromised astrocyte viability in vitro. The ACSA‐1 antibody is not only a valuable tool to identify and track astrocytes by immunostaining, but also provides the possibility of separation and further analysis of pure astrocytes. © 2012 Wiley Periodicals, Inc.