In order to understand gene regulation by glucocorticoids, it is pivotal to know how the major transactivation domain AF1 of the glucocorticoid receptor (GR) functions. Located in the N-terminal region of the GR, AF1 is quantitatively important for transcriptional regulation, but only in recent years have we begun to understand how AF1 works. This is in part due to the fact that the recombinant AF1 (rAF1) peptide exists as a random ensemble of conformers. Algorithms that predict structure support the view that AF1 is also not well ordered in the holo-GR, and the properties of the amino acids in AF1 suggest that it is intrinsically disordered. However, it is generally believed that intrinsically disordered sequences of the GR AF1 must achieve one or more ordered conformation(s) to carry out transactivation activity. Based on our previous published work and available literature, we hypothesize that a confluence of effects that operate under physiological conditions cause functionally active conformation(s) to form in AF1. We have shown that when rAF1 is incubated in increasing concentrations of a naturally occurring osmolyte trimethylamine-N-oxide (TMAO), the peptide folds into functionally active conformation(s) that selectively binds several critical coregulatory proteins. Because cells contain various organic osmolytes whose effects may be cumulative, and in light of cell-specific effects of GR AF1 action, we tested whether it can be folded by other natural organic osmolytes representative of three classes: certain amino acids (proline), methylamines (sarcosine), and polyols (sorbitol). The osmolyte-induced folding of rAF1 shows greatly increased affinity for specific binding proteins, including TATA box-binding protein (TBP), CREB-binding protein (CBP), and steroid receptor coactivator-1 (SRC-1). Consistent with theory and published data with other proteins, our results show that different osmolytes have differential effects on rAF1 folding. The cell-specific functions of the GR AF1--and by extension the AF1s of other nuclear hormone receptors--may in part be affected by the presence and concentrations of particular osmolytes within a particular cellular environment.