The addition of molybdate to intact or homogenized cells of the endometrial adenocarcinoma line HEC-1 during incubation with [3H]estradiol ([3H]E2) at 4 C causes substantial increases in cytoplasmic E2 binding levels. A similar effect can be observed in homogenates of normal human endometrium. These effects of molybdate appear to involve activation of E2-binding sites. Fractionation of the homogenates and recombination of different fractions revealed that activation of specific E2-binding sites by by MoO4= requires cytosolic factors as well as factors associated with the cell membrane. In homogenates of neoplastic cells (HEC-1) and normal endometrium, the addition of ATP, GTP, or cGMP was also found to increase E2 binding to levels as high as those obtained by the addition of MoO4=. In contrast, the addition of cAMP was found to lower specific E2 binding levels and to counteract the effects of MoO4=, ATP, GTP, and cGMP. Levels of intracellular cAMP and cGMP can change rapidly in cells in culture. Since cGMP causes E2 binding levels to increase while cAMP causes them to decrease, changes in the levels of these two cyclic nucleotides may explain the fluctuation in concentrations of specific estrogen binders that we have previously reported to occur in cultured endometrial cells.