Hepatic nuclear factor-4alpha (HNF-4alpha) controls the expression of genes encoding proteins involved in lipid and carbohydrate metabolism. Fatty acyl-CoA thioesters have recently been proposed to be naturally occurring ligands of HNF-4alpha and to regulate its transcriptional activity as function of their chain length and degree of unsaturation (Hertz, R., Magenheim, J., Berman, I., and Bar-Tana, J. (1998) Nature 392, 512-516). However, the apparent low affinities (microm K(d) values) obtained with a radiolabeled fatty acyl-CoA ligand binding assay raised questions regarding the physiological significance of this finding. Furthermore, it is not known whether interaction with fatty acyl-CoA alters the structure of HNF-4alpha. These issues were examined using rat recombinant HNF-4alpha ligand-binding domain (HNF-4alphaLBD) in conjunction with photon counting fluorescence and circular dichroism. First, fluorescence resonance energy transfer between HNF-4alphaLBD tryptophan (Trp) and cis-parinaroyl-CoA yielded an intermolecular distance of <or=42 A, thus pointing to direct molecular interaction rather than nonspecific coaggregation. Second, quenching of HNF-4alphaLBD intrinsic Trp fluorescence by fatty acyl-CoAs (e.g. pamitoyl-, stearoyl-, linoleoyl-, and arachidonoyl-CoAs) yielded a single binding site with K(d) values of 1.6-4.0 nm. These affinities were 2-3 orders of magnitude higher than those previously derived by radiolabeled fatty acyl-CoA ligand binding assay. Third, binding of fatty acyl-CoAs was specific as the binding affinities of the respective free fatty acids or free CoA (K(d) values of 421-742 nm) were significantly lower. Fourth, circular dichroism demonstrated that the HNF-4alphaLBD secondary structure was significantly and differentially altered by fatty acyl-CoA binding. The opposite effects of saturated versus polyunsaturated fatty acyl-CoAs on HNF-4alpha LBD secondary structure correlated with their opposite regulatory effects on HNF-4alpha function. Fifth, the CoA thioesters of some hypolipidemic peroxisome proliferators bind with high affinity (K(d) values as low as 2.6 nm) to HNF-4alpha LBD, thus indicating that HNF-4alpha may serve as target for these drugs. In summary, these data demonstrate for the first time high affinity binding to HNF-4alpha of fatty and xenobiotic acyl-CoAs in the physiological range, resulting in significantly altered HNF-4alpha conformation.