Type II estradiol binding sites characterized by lower affinity and higher capacity than type I receptor sites have been described in rat uterine nuclei. These sites appeared to be dependent on estrogen stimulation. Reducing agents prevented estradiol binding to these sites. In the present study, the situation prevailing in adult rats (Ad) was studied and compared to ovariectomized (Ox) and ovariectomized estrogen prestimulated rats (OxPS). Nuclear precipitate from Ad, Ox and OxPS rats were incubated with tritiated estradiol (E2(3)H) in the presence and in the absence of mercaptoethanol as reducing agent. In the presence of mercaptoethanol, saturation was attained at E2(3)H concentrations above 16 nM. In the absence of reducing agents, a secondary binding was observed in Ad and OxPS which was not saturated at E2(3)H levels up to 80 nM. Non-specific binding obtained with paired aliquots containing 100-fold excess of DES as competitor was not linear but showed a saturation profile, distorting the saturation curve of the specific sites, obtained by subtracting non-specific from total E2(3)H binding. Increasing DES concentrations up to 10,000 nM did not allow to reach complete exchange with E3(3)H ligand bound to specific sites, preventing measurement of binding sites concentration. Incubation of nuclear fractions with increasing concentrations of E2(3)H (up to 6,000 nM) gave a saturation curve with a linear kinetics above 1-2,000 nM, which represented saturation concentration of the specific sites. From this, non-specific and specific moieties could be estimated. Binding capacity of specific sites was of the order of 50-80 pmol uterus. Half saturation was attained between 300 and 600 nM E2(3)H, which approximated the Kdiss of these sites, at variance with the Kdiss of 15-30 nM originally reported for type II binding sites. In conclusion, these results show that secondary binding sites were present in uterine nuclei of Ad and OxPS rats. Binding capacity was about 30-fold higher than that of type I sites. Affinity was however very low, and casts some doubt on the role of these sites as active estradiol binders in physiological situations. Their increase under the influence of estrogen may however be related to some as yet undetermined role.