Cytoplasmic estrogen receptor (CER) activation and nuclear estrogen receptor (NER) forms have been examined in normal virgin and lactating mammary gland by 1) measuring [3H]estradiol-receptor dissociation kinetics, and by 2) sucrose density gradient analysis of sedimentation behavior on high salt gradients. The data show two forms of CER in both virgin and lactating mammary gland: 1) a fast dissociating-nonactivated form which sediments at 4 S, and 2) a slow dissociating-activated form which sediments at 5 S. The 5 S slow dissociating estrogen receptor (ER) is the predominant form found in the nucleus. CER are activated by exposure to elevated temperature, high salt concentration, dilution, or ammonium sulfate fractionation. This is demonstrated by an increase in the proportion of the slow dissociating, 5 S ER form. Sodium molybdate inhibits activation and prevents heat- or ammonium sulfate-induced increase in the amount of the slow-dissociating CER as well as the 4 S to 5 S increase in sedimentation coefficient. We also found that cytoplasmic and nuclear 5 S ER forms were susceptible to 1) cold-induced dissociation, and to 2) degradation by nuclear protease activity. Thus, whereas mammary gland CER activation and NER forms described herein are very similar to those reported for other estrogen target tissues, the experimental conditions required to demonstrate the activated-nuclear form of ER differ substantially from those reported for uterine tissue. These findings may be important for assessing differences in ER forms in certain disease states such as mammary neoplasia. Previously we have shown that lactating mammary gland, in contrast to virgin mammary gland, is not responsive to estrogen. Having compared CER and NER forms and receptor activation in virgin vs. lactating mammary gland, we find no differences between ER in these two tissue states that can explain the absence of responsiveness to estrogen during lactation.