To determine whether sex steroids play any role in the increased morbidity associated with acute symptomatic hyponatremia in menstruant females, we studied the actions of estradiol, progesterone, and testosterone on regulatory volume decrease (RVD) of brain astrocytes in culture. To determine intracellular space with the use of 3-O-[methyl-D-3H] glucose, cells were cultured in media containing either estradiol or progesterone. Those treated with ouabain were unable to regulate volume normally, whereas testosterone-treated cells displayed normal RVD. After 15 s of hypotonic exposure, control cell volume and 100 nM testosterone-treated cell volume increased by 26 and 31%, respectively. Cell volume in control cells changed from 1.74 +/- 0.24 to 2.41 +/- 0.28 microliters/mg protein. At the same time, cells treated with either 10 nM estradiol or 10 nM progesterone significantly (P < 0.01) increased their volume by 129 and 90%, respectively. Both the antiestrogen agent tamoxifen and the antiprogesterone agent mifepristone (RU-486) blocked the effects of estradiol and progesterone. The Na-K-ATPase pump, which plays an important role in cell RVD, was significantly (P < 0.03) inhibited by 32 and 21% in estradiol- and progesterone-treated cells, but significantly (P < 0.001) stimulated (49%) by testosterone treatment. Taken together, these results provide a possible explanation for the increased morbidity associated with acute symptomatic hyponatremia in menstruant females.