Previous studies have documented that cultured Schwann cells require serum-containing medium to respond maximally to mitogens. We now report that Schwann cells are able to proliferate to a mitogenic response in a serum-free defined medium termed oligodendrocyte defined media (ODM). Glucocorticoids are the essential component of ODM which allow Schwann cell proliferation in the serum-free medium. Charcoal treatment of the fetal calf serum decreases the mitogenic potency of the axolemma-enriched fraction (AEF) by 50%. The addition of 2 microM hydrocortisone to charcoal-treated fetal calf serum restores 75% of the lost mitogenicity. These observations are consistent with the view that glucocorticoids present in fetal calf serum are potent co-mitogens essential for AEF-induced Schwann cell proliferation. The synthetic glucocorticoid, dexamethasone, is a more potent co-mitogen than hydrocortisone, with a maximal effect at concentrations less than 10 nM. In contrast, other steroids including aldosterone, progesterone, testosterone, and 17 beta-estradiol have no effect on enhancing the mitogenic response of Schwann cells to the AEF. The glucocorticoid antagonists RU 486 and dehydroepiandrosterone (DHEA), but not the antiestrogenic compound tamoxifen, block AEF-induced Schwann cell proliferation. These results suggest that glucocorticoid-induced Schwann cell proliferation is mediated through a glucocorticoid receptor (GR) mechanism. We detected immunoreactivity to the GR in the cytoplasm, but not in the nuclei of Schwann cells grown in ODM lacking dexamethasone. The addition of 100 nM dexamethasone to these cultures resulted in immunoreactivity in the nucleus. This data suggests that glucocorticoids working through the GR are potent co-mitogens for Schwann cell proliferation.