We describe the use of immunopanning to purify rodent astrocytes. Immunopanning of astrocytes permits the prospective isolation of astrocytes from the rodent brain. Prospective isolation refers to the direct selection of cells without multiple steps that extend over a few days, thereby permitting the selection of a representative proportion of the astrocytes from the cortex. Because immunopanning is a very gentle process, the cells are viable at the end of the preparation and can be cultured in a serum-free medium containing heparin-binding EGF-like growth factor (HBEGF), the critical survival factor of astrocytes in vitro, for at least 2 wk. This protocol was initially established and verified with rats, but modifications for the purification of mouse cells are also described here.