We investigated the calf uterine estrogen receptor's interaction with [3H]estriol and [3H]estrone and found that the receptor's cooperative binding is markedly dependent upon temperature and receptor concentration. With increasing temperature, the intensity of the estrogen receptor's cooperativity decreases: at 0 degrees C the Scatchard plot was convex and the Hill coefficient was 1.58 +/- 0.04 (n = 5), while at 30 degrees C the Scatchard plot approached linearity and the Hill coefficient was 1.03 +/- 0.02 (n = 3) for the binding of [3H]estriol with a receptor concentration of 10 to 12 nM. With increasing receptor concentration, the receptor's cooperativity gradually increased. At a receptor concentration of 1 nM, at 25 degrees C, with [3H]estriol the Hill coefficient was low, 1.03 +/- 0.02 (n = 3), while at approximately intracellular receptor concentration (20 nM) the Hill coefficient increased to 1.20 +/- 0.04 (n = 4). Similar results were observed with [3H]estrone. The cooperative interaction of the estrogen receptor with [3H]estriol or [3H]estrone is reversible and dependent upon temperature. The van't Hoff analysis of the apparent dissociation constant of the [3H]estrone- and [3H]estriol-receptor complexes indicates that a transition in the receptor's binding mechanism occurs at 15 degrees C. Therefore, measurements of ligand interactions with the estrogen receptor at 0 to 15 degrees C, may not reflect the binding mechanism of the receptor at more biologically relevant temperatures. The reduced, positively cooperative interactions of [3H]estriol and [3H]estrone with the estrogen receptor at 30 degrees C, in comparison with estradiol, decrease the presence of the activated receptor, which correlates with the partial agonist-antagonist activities of estriol and estrone observed in vivo.