A variety of anti-estrogenic substances have been examined for their direct and indirect effect on the binding of [ 3H]-estradiol with uterine cytoplasmic receptors. A distinct separation of the compounds into 2 classes of inhibitors was observed. The non-steroidal anti-estrogens and the C 19 steroids, except Norgestrel, interacted with cytoplasmic binding sites. Testosterone, norprogesterone, the C 21 steroids and Norgestrel did not suppress binding in vivo or in in vivo. These results demonstrate that interference with estradiol binding to uterine cytoplasmic receptor sites is not essential for the inhibition of estrogen responses by some of the most potent anti-estrogenic substances. [ 3H]-Estradiol uptake was enhanced by low doses of compounds which at higher doses inhibited uptake. This observation demonstrates the need for a dose response when examining this aspect of competition. Direct interaction with uterine cytoplasmic binding sites in a cell-free system was observed with agents which reduce [ 3H]-estradiol uptake in vivo. After in vivo injection of estradiol, uterine cytoplasmic binding capacity for [ 3H]-estradiol is reduced within the first 5 h, followed by a recovery phase. The dynamics of this process appear to be influenced by the dose of estrogen administered. Interference with the recovery phase is suggested as a possible mechanism of inhibition for compounds having no effect on cytoplasmic binding of estrogen.