Digitalis preparations have a weak estrogenic effect in man. The data in animals are equivocal. We have studied.the biologic effect of both digitoxin and digoxin on the rat uterus in vivo and the interaction of these drugs with the rat uterus estrogen receptor in vitro. Digitoxin and estradiol significantly increased the uterine weight of immature rats, while digoxin did not. The interaction of digitoxin and digoxin with the rat uterus estrogen cytosol receptor was studied using protamine sulfate precipitation and dextran-coated charcoal (DCC) assays. Both methods gave a Kd for the estradiol-receptor interaction between 0.8-3.1 X 10(-9) M (n = 20). Digitoxin at concentrations of 0.5-2.0 X 10(-6) M significantly inhibited the binding of estradiol to the specific or saturable binding sites with minimal inhibition of hormonal binding to nonspecific sites. The binding was competitive with a calculated Ki for digitoxin of 5.2-7.8 X 10(-7) M (n = 18). Digoxin failed to inhibit estradiol binding to the receptor protein in vitro. We conclude that digitoxin probably acts directly as a weak estrogen and that this effect probably explains the estrogen-like side effects seen with digitoxin therapy in man.