Glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gene requires a glucocorticoid response unit (GRU) comprised of two non-consensus glucocorticoid receptor (GR) binding sites, GR1 and GR2, and at least three accessory factor elements (gAF1-3). DNA-binding accessory proteins are commonly required for the regulation of genes whose products play an important role in metabolism, development, and a variety of defense responses, but little is known about why they are necessary. Quantitative, real time homogenous assays of cooperative protein-DNA interactions in complex media (e.g. nuclear extracts) have not previously been reported. Here we perform quantitative, real time equilibrium and stopped-flow fluorescence anisotropy measurements of protein-DNA interactions in nuclear extracts to demonstrate that GR binds to the GR1-GR2 elements poorly as compared with a palindromic or consensus glucocorticoid response element (GRE). Inclusion of either the gAF1 or gAF2 element with GR1-GR2, however, creates a high affinity binding environment for GR. GR can undergo multiple rounds of binding and dissociation to the palindromic GRE in less than 100 ms at nanomolar concentrations. The dissociation rate of GR is differentially slowed by the gAF1 or gAF2 elements that bind two functionally distinct accessory factors, COUP-TF/HNF4 and HNF3, respectively.