The presence of an estrogen-inducible progesterone receptor was demonstrated in primary cultures of newborn rat glial cells by biochemical and immunohistochemical techniques. The progesterone receptor (PR) was measured 3-4 weeks after primary culture in estradiol-containing or control medium. Cells were labeled with the synthetic progestin [3H]R5020 followed by ultracentrifugation analysis of the cellular extracts. A "9 S" PR was observed in the cytosol and a "4-5 S" PR was found in the nuclear high salt, tungstate ions containing extract of estradiol-treated cells. When the antiprogestin [3H]RU486 was used instead of [3H]R5020 as a ligand, a 9 S PR was also found in the cytosol, but a nonactivated "8.5 S" receptor complex was identified in the high salt nuclear fraction in presence of tungstate ions. The levels of PR, as measured by whole cell assay, were significantly increased when glial cells were cultured in the presence of 50 nM estradiol, as compared to nonestradiol-treated controls. The estrogen induction of PR was suppressed by the antiestrogen tamoxifen, but tamoxifen by itself had no effect on PR concentration. When the glucocorticosteroid receptor and PR were measured in parallel after estradiol treatment of the same primary culture, only the levels of PR were increased. The PR was visualized inside glial cells by immunohistochemical studies with a monoclonal antibody specific for the B-form of PR (KC 146), which was recognized by fluorescein-linked or biotinylated secondary antibodies. Strong staining was observed in estradiol-treated cultures, when compared to a weaker staining in control cultures. This is the first demonstration of PR in rat glial cells, and we present evidence of its induction by estradiol in primary cultures.