Astrocytes have been prepared from adult rat cortex, cerebellum, and striatum, using a modification of the McCarthy-DeVellis (J Cell Biol 85:890, 1980)method. The cultures consist of 99% type 1 polygonal astrocytes, which divide more slowly than cells from newborn animals. One day after preparing the cultures, 90% of the cells are glial fibrillary acidic protein (GFAP)-positive and 80% are tin-positive by immunohistochemical staining, suggesting that they are present de novo and not derived from precursor cells. The astrocytes from adult brain respond to an elevation of intracel-lular cyclic AMP, following treatment with forskolin, by becoming more stellate in shape and putting out fine ramified processes. They contain the same amount of GFAP per mg protein, measured by immunoblot, as cells from newborn animals. These cultures thus offer the possibility of comparing the biochemical properties of astrocytes derived from adult animals with those from newborn animals, or with cultures of reactive astrocytes isolated from lesioned brain.