In addition to suggesting that fatty acids are endogenous ligands, our recent crystal structure of HNF-4α showed an unusual degree of structural flexibility in the AF-2 domain (helix α12). Although every molecule contained a fatty acid within its ligand binding domain, one molecule in each homodimer was in an open inactive conformation with α12 fully extended and colinear with α10. By contrast, the second molecule in each homodimer was in a closed conformation with α12 folded against the body of the domain in what is widely considered to be the active state. This indicates that although ligand binding is necessary, it is not sufficient to induce an activating structural transition in HNF-4α as is commonly suggested to occur for nuclear receptors. To further assess potential mechanisms of activation, we have solved a structure of human HNF-4α bound to both fatty acid ligand and a coactivator sequence derived from SRC-1. The mode of coactivator binding is similar to that observed for other nuclear receptors, and in this case, all of the molecules adopt the closed active conformation. We conclude that for HNF-4α, coactivator rather than ligand binding locks the active conformation.