In previous reports we challenged the concept that uterine nuclei of rats contain two forms of estrogen receptors, one salt extractable and the other salt resistant. Although it is likely that a certain fraction of the nuclear bound receptor-estrogen complex exists as a ternary high-affinity acceptor-receptor-estrogen complex, current salt extraction procedures do not allow discrimination between receptor-17beta-estradiol complexes associated with high-affinity and low-affinity nuclear binding sites. Recent reports suggested that the DNA-intercalating agent ethidium bromide selectively extracted those sites that appeared to be salt resistant. In view of contradictory reports to this effect, we have attempted to clarify this issue. The data presented indicate that ethidium bromide is not a useful tool for the identification of a specific class ("salt-resistant") of nuclear binding sites for receptor-17beta-estradiol complexes. This conclusion is based on measurement of nuclear bound receptor-17-beta-estradiol following KCl and/or ethidium bromide extraction by using both the direct assay and the nuclear exchange assay.