Estrogen receptors (ER) alpha and beta bind estradiol (E2) and other estrogenic ligands with different affinities. To measure the rate of E2 association with ERa and ERbeta, we employed tetrahydrocrysene ketone (THCK), a fluorescent ligand that is an agonist with ERalpha and an antagonist with ERbeta. We report that THCK binds E2-liganded and unliganded ERalpha and ERbeta, indicating a THCK binding site(s) other than the E2 binding pocket. THCK fluorescence was greater for ligand-occupied ERbeta than ERalpha, suggesting differences in the microenvironment of the THCK binding site(s). THCK fluorescence was also significantly greater for E2-, 4-hydroxytamoxifen-, and tamoxifen aziridine-liganded versus unliganded ER, allowing calculations of E2 association rate constants (ka) of 7.60 +/- 0.75 and 5.12 +/- 0.30 x 10(5) M(-1) s(-1) for E2-ERalpha and E2-ERbeta, respectively. THCK did not affect ERalpha binding to estrogen response element (ERE) DNA, but decreased ERbeta-ERE binding. We conclude that THCK binding site(s) on ERalpha versus ERbeta are different and important for ER function.