Primary cultures of glial cells from 2-day-old rat cerebellum were used to examine the growth control properties of steroid hormones. Immunocytochemical staining with antiglial fibrillary acidic protein (anti-GFAP) demonstrated that the cultures were highly enriched for astrocytes (90%). In an effort to avoid the potential influence of serum-borne steroids, cultures were switched from serum-supplemented to serum-free, chemically defined medium prior to experimentation. Assays for DNA synthesis used [3H]thymidine incorporation with either liquid scintillation counting of TCA-insoluble material or light microscopic autoradiography. Glial cells grown in serum-free, chemically defined medium (F12 basal medium supplemented with putrescine, selenium, insulin, transferrin, and BSA) replicated their DNA to a limited extent even in the absence of serum mitogens. When the glial cells were shifted to defined medium supplemented with various steroid hormones (corticosterone, dexamethasone, hydrocortisone, 17 beta-estradiol, progesterone, or testosterone) at a concentration of 10(-7) M, it was found that the glucocorticoids, corticosterone and dexamethasone inhibited synthesis of DNA by 49.6 and 56.9%, respectively. Hydrocortisone, another glucocorticoid, caused only a small reduction in DNA synthesis. The growth-controlling activity of the glucocorticoids was dose-dependent with concentrations of 10(-7) -10(-6) M showing maximal effect on DNA synthesis. These results suggest that physiological concentrations of glucocorticoid hormones may exert negative control over DNA synthesis of glial cells in the developing or injured central nervous system.