The effect of increasing ionic strength on the efficacy of the charcoal adsorption assay for estrogen receptors has been examined. Cytosol prepared from immature rat uteri was exposed to variable concentrations of KCl and the saturable or "specific" binding of [3H]estradiol was measured by charcoal and hydroxylapatite adsorption, gel filtration, and/or density gradient centrifugation using excess diethylstilbestrol to correct for non-saturable or "non-specific" binding. As the concentration of KCl was increased, the number of estradiol binding sites as measured via charcoal adsorption decreased in proportion to the conversion of receptor from a low salt (8S) to a high salt (4S) complex. This "stripping" of [3H]estradiol from the 4S receptor species was both time- and charcoal concentration-dependent. It is concluded that the charcoal adsorption procedure quantitatively assesses the binding of [3H]estradiol to low salt, 8S receptor species but is not the assay of choice for salt-extracted or salt-treated receptors in view of the potential for artifactually low estimates of receptor number. Low values for receptor number under high salt conditions may result from exposure of the binding site of the estrogen receptor to charcoal.