An improved assay for measuring ligand binding to extracted nuclear type II estrogen binding sites which involves preimmobilization on glass fiber filters is described. At least two classes of specific estrogen binding sites have been demonstrated in rat uterus as well as in a variety of other tissues and species and have been designated as type I and type II. Although the endogenous ligand to the type II binding site has recently been identified as methyl p-hydroxyphenyllactate (MeHPLA), tritiated estrogens are generally used for radiolabeling this site due to the susceptibility of MeHPLA to enzymatic hydrolysis in in vitro assays. After extracting the type II site from the nuclear matrix, ligand binding and protein stability appear to be significantly enhanced by first immobilizing the site on an artificial matrix, such as hydroxylapatite, before incubating with radiolabeled ligand. Immobilization of the extracted site on glass fiber filters results in higher specific binding and lower nonspecific binding when compared to hydroxylapatite and a number of other immobilization matrices. The glass fiber ligand exchange procedure for measuring type II binding can also be performed on smaller samples and requires less time than other methods. Type II sites are significantly stabilized when immobilized on glass and exhibit sigmoidal binding curves when incubated with increasing concentrations of [3H]estradiol and [3H]estrone and display inhibition data characteristic of that observed using more traditional assays.(ABSTRACT TRUNCATED AT 250 WORDS)