To investigate the production of angiotensinogen by the brain, primary cultures were prepared from the brains of one-day-old rats. Two to four weeks after plating, they were transferred to serum-free medium. The cultures, which contained approximately 15% neurons, 80% astroglia and 5% other types of cells, produced angiotensinogen at a steady rate for three to four days in serum-free medium. Cultures prepared from subcortical tissue produced more angiotensinogen than cultures prepared from cerebral cortical tissue. Angiotensinogen mRNA was also identified in those cultures. Forskolin treatment had no effect on angiotensinogen production. Astroglia-enriched cultures that contained no identifiable neurons also produced angiotensinogen and its mRNA. Astroglial cells from hypothalamus and thalamus produced more of both than astroglial cells from the cerebral cortex. In situ hybridization histochemistry on sections of the hypothalamus of adult male rats showed a diffuse distribution of cells containing angiotensinogen mRNA that was more consistent with a glial than a neuronal distribution. The data indicate that most if not all of the angiotensinogen in rat brain is produced by astrocytes.