PIP: The total binding capacity of the cell receptors and not only the fraction required to elicit physiological response was detected and it is shown that specific receptors filled by endogenous estradiol were detected along with the unfilled sites. Changing the temperature of the incubation media altered intracellular distribution of bound estradiol. Late nucleolar retention of estradiol was shown after its release from nuclear protein. This unexpected finding may be a key event. Tumor tissues were obtained from 40 primary breast cancers, 2 endometrial carcinomas, and 1 lung and 1 gastric cancer; 2 normal human spleens, 2 mouse livers, and 1 breast from a 5-month pregnant women were processed. Tissue samples were transferred to a cold flask containing phosphate-buffered balanced salt solution. Further details of preparation are given to show typical estradiol-target cells containing variable amounts of specific estradiol-receptors. For immunofluorescence staining and indirect technique was used to show the in situ estradiol localization. The fluorescence staining showed 3 patterns. Cells incubated in the cold mostly displayed a bright, homogeneously diffuse fluorescence of the cytoplasm with the nuclear area unstained. There were some negative cells. The 2nd pattern of fluorescence was shown by those incubated at room temperature. Cytoplasmic staining was more marked and nuclear areas showed bright-fluorescent light stippling. A 3rd staining pattern was seen after slow postincubation warming up to 37 degrees C when increased nuclear staining occurred. Some cells did not show any nuclear labeling despite the clear cytoplasmic fluorescence. Preparations of human spleen, nontarget tumors, or mouse liver had no fluorescent cells. All fluorescent stainings were prevented by preincubation and washing in media containing Nafoxidine and N-ethylmaleimide. Control experiments confirmed the immune specificity of the detection of estradiol. Breast tissue cells from a pregnant women showed moderate cytoplasmic and faint nuclear fluorescence before incubation with estradiol, after which cytoplasmic staining was reinforced. Cells from the premenstrual beast cancers sometimes showed fluorescence without exposure to estradiol.