Crude nuclear preparations from the rat uterus and the MXT mouse mammary tumor contain endogenous nucleases whose activity results in the loss of estrogen-binding sites from these fractions due to DNA breakdown. Although this is not a significant problem for studies in the uterus, the majority of the receptors present in nuclei from the tumor were lost from the particulate fraction under the standard conditions for the [3H]estradiol exchange assay. The addition of 5 mM Ca++ to either of these nuclear preparations resulted in the aggregation of the released estrogen receptors with particulate components and, hence, a total inhibition of receptor release into the soluble (wash) fractions. Ca++ had no effect on the affinity of the receptors for [3H]estradiol, although in some cases it slowed the overall rate of binding. The addition of Ca++ to the nuclear estrogen exchange assay may therefore prove to be a simple yet powerful aide in studies on the potential hormonal responsiveness of human and experimental mammary tumors.