The glucocorticoid receptor initiates the cellular response to glucocorticoid steroid hormones in vertebrates. Co-regulator proteins dock to the receptor in response to hormone binding and potentiate the transcriptional activity of the receptor by modifying DNA and recruiting essential transcription factors like RNA polymerase II. Hormones and co-regulators bind at distinct sites in the ligand binding domain yet function cooperatively to mediate transcriptional control. This study reveals and quantifies energetic coupling between two binding sites using purified components. Using a library of peptides taken from co-regulator proteins, we determine the pattern of co-regulator binding to the glucocorticoid receptor ligand binding domain. We show that peptides from co-regulators differ in their effects on hormone binding and kinetics. Peptides from DAX1 and SRC1 bind with similar affinity, but DAX1 binding is coupled to hormone binding, and SRC1 is not. Mechanistic details of co-regulator binding and coupling to the hormone binding pocket are uncovered by analysis of properties endowed by mutation of a key residue in the allosteric network connecting the sites.