The rate of dissociation of recombinant, purified human estrogen receptor α (ERα) from a fluorescein-labeled DNA containing the consensus vitellogenin ERE sequence (F-vitERE) was determined in real time using fluorescence anisotropy. The complex of estradiol-occupied ERα with F-vitERE had an apparent dissociation rate of 1.48±0.06×10 −2 s −1 and a half-life of 46.6 s at room temperature. The dissociation rate was characterized by a single exponential decay, suggesting that ER dissociates from the DNA as a preformed dimer, rather than as two individual monomers. The association rate of estradiol-occupied ERα for the F-vitERE was calculated as 7×10 6 M −1 s −1 based on the dissociation rate measured and previous determinations of the equilibrium dissociation constant (K d ) in similar assay conditions (Ozers et al., 1997). In buffer containing various concentrations of salt, the rate of dissociation of estradiol-occupied ERα from F-vitERE was accelerated by increasing salt concentrations. Compared to estradiol-occupied ERα, the rate of dissociation of unoccupied ERα from the F-vitERE was very similar, indicating that estradiol occupancy does not affect the dissociation rate of ERα from the ERE.