A rat hepatoma cell line in tissue culture (HTC cells) was treated with hypophyseal extracts from adult male and female rats. Cell homogenates were then assayed for steroid metabolizing enzymes using 4-androsten-3,17-dione as substrate. The major products were the 5 alpha-reduced derivatives (5 alpha-androstane-3,17-dione, androsterone, and epiandrosterone). When the cells were grown in the presence of female hypophyseal extract the apparent activity of the 5 alpha-reductase increased markedly, whereas treatment with male hypophyseal extract was without effect. Treatment with female hypophyseal extract resulted in a marked decrease in the apparent Km for 5 alpha-reductase from 667 +/- 102 to 99 +/- 4 muM in addition to a decrease in the apparent Vmax from 67 +/- 12 to 46 +/- 2 pmol of product/min per mg of protein. A logarithmic dose-response was obtained with female hypophyseal extract. Treatment of the HTC cells with purified rat hypophyseal follicle-stimulating hormone, luteinizing hormone, growth hormone, thyrotropic hormone, and prolactin had only marginal effects on 5 alpha-reductase activity. Crude female hypophyseal extracts were at least 6-fold more potent than any of the standard hormone preparations and at least 250-fold more potent than male hypophyseal extracts when based on activity per mg of pituitary tissue. Chromatography of crude female hypophyseal extracts on Sephadex G-25 indicated that the factor was of high molecular weight. The identity of this activity with a hypophyseal "feminizing" factor is postulated.