Rat uterine nuclei contain two types of estrogen binding sites (I and II). Type I is the classical high-affinity, low-capacity binding component, while Type II has lower affinity and higher capacity. Investigation of the presence and number of estrogen-binding proteins in isolated uterine nucleoli, and the possible role of the estrogen-binding protein(s) in the stimulation of nucleolar RNA synthesis was undertaken. Isolated uterine nucleoli contain a large number of lower-affinity binding sites (Type II) but are devoid of a significant number of high-affinity binding protein(s) (Type I). Following in vivo treatment with estradiol the number of detectable Type II estradiol-binding sites in isolated uterine nucleoli increased with time of estrogen treatment, peaking between 16 and 24 h after hormone administration and gradually decreasing to control levels between 48 and 72 h. The estrogen-activated binding activity but not the basal activity is sensitive to dithiothreitol and insensitive to beta-mercaptoethanol during the in vitro assay, suggesting that important disulfide bonds may be involved in the estrogen-induced nucleolar binding sites. The in vivo activation of nucleolar estradiol-binding sites exhibits steroid specificity. Data indicate that a strong correlation exists between activation of uterine nucleolar transcriptional and estradiol-binding activities.