The measurement of E2 receptor (E2R) in human breast cancer cytosol is significantly influenced by conditions usually employed in the dextran-coated charcoal assay. The incubation time and temperature have an influence on the rate of binding and stability of the receptor. Since lower temperatures preserve the integrity of the receptor, a 2 hr incubation at 4 degrees was selected as the standard incubation procedure. These conditions allow for the detection of at least 80% of the E2R. With supernatants from high-speed centrifugation of HBT biopsies or the human breast cancer cell line MCF-7, reducing agents increased the apparent E2R binding in the order: DTT greater than G-SH greater than MTG. The maximum enhancement of specific E2R binding by a given thiol agent was dependent on its concentration in the incubation medium. The optimum DTT level (7.5 mM) for MCF-7 cell homogenization and cytosol equilibration with tritiated E2 increased E2R to two times control (no DTT). For the HBT 150,000 g supernatant, 1 mM DTT was required to optimize the E2R quantitation. The duration of the dextran-coated charcoal extraction of the cytosol-[3H]E2 incubation had no effect on the level of E2R up to 21 hr. Minimum levels of nonspecific binding of [3H]E2 could be obtained after 4 hr extraction. Maximum depletion of specific [3H]E2 binding could be obtained by adding between 200- and 1000-fold molar excess of unlabeled E2. Greater amounts of unlabeled steroid displaced the radioactive E2 from the dextran-coated charcoal, thereby artifactually increasing the apparent nonspecific binding. This phenomenon may be overcome by utilizing more dextran-coated charcoal in the extraction. However, there was a 9% loss of specifically bound [3H]E2 per milligram of dextran-coated charcoal (1:10 dextran to charcoal by weight) when the cytosol protein was below 90 microgram per incubation. Supplementation with 200 microgram or more albumin per incubation prevented this loss. The dextran:charcoal ratio also prevented E2R loss in the order: 1:1 greater than 1:10 greater than 1:100. One milligram of dextran-coated charcoal (1:10) has the capacity to adsorb 0.3 to 0.4 microgram of free E2. Other unlabeled competitors are capable of displacing [3H]E2 on the receptor. Although DES was as effective as E2, U11,100A and estrone were inefficient competitors. It appeared that the levels of these two estrogen analogues required to maximally displace [3H]E2 on receptor also eluted labeled E2 from the dextran-coated charcoal. DES, however, was unable to displace significant quantities of the [3H] E2 from dextran-coated charcoal even at a molar excess of 50,000:1.