Dietary fatty acids specifically modulate the onset and progression of various diseases, including cancer atherogenesis, hyperlipidaemia, insulin resistance and hypertension, as well as blood coagulability and fibrinolytic defects; their effects depend on their chain length and degree of saturation. Hepatocyte nuclear factor-4α (ref.8) (HNF-4α) is an orphan transcription factor of the superfamily of nuclear receptors and controls the expression of genes (reviewed in ref. 9) that govern the pathogenesis and course of some of these diseases. Here we show that long-chain fatty acids directly modulate the transcriptional activity of HNF-4α by binding as their acyl-CoA thioesters to the ligand-binding domain of HNF-4α. This binding may shift the oligomeric-dimeric equilibrium of HNF-4α or may modulate the affinity of HNF-4α for its cognate promoter element, resulting in either activation or inhibition of HNF-4α transcriptional activity as a function of chain length and the degree of saturation of the fatty acyl-CoA ligands. In addition to their roles as substrates to yield energy, as an energy store, or as constituents of membrane phospholipids, dietary fatty acids may affect the course of a disease by modulating the expression of HNF-4α controlled genes.