Very-long-chain fatty acids (VLCFA) and branched-chain fatty acids (BCFA) are potent inducers of the peroxisome proliferator-activated receptor PPARalpha, a nuclear receptor that enhances transcription of peroxisomal enzymes mediating beta-oxidation of these potentially toxic fatty acids. However, it is not known whether the respective free fatty acids or their activated metabolites, i.e., CoA thioesters, (i) are the endogenous high-affinity PPARalpha ligands, (ii) alter PPARalpha conformation, and (iii) alter recruitment of coregulatory proteins to PPARalpha. As shown by quenching of PPARalpha intrinsic amino acid fluorescence, PPARalpha exhibited high affinity (3-29 nM Kds) for the CoA thioesters of the common (C20-C24) VLCFA. In contrast, with the exception of arachidonic acid (Kd = 20 nM), PPARalpha only weakly bound the VLCFA. PPARalpha also exhibited higher affinity for the CoA thioesters of BCFA (phytanoyl-CoA, pristanoyl-CoA; Kds near 11 nM) than for the respective free branched-chain fatty acids. As shown by circular dichroism, the high affinity VLCFA-CoA and BCFA-CoA strongly altered PPARalpha conformation. Likewise, the high affinity VLCFA-CoA and BCFA-CoA altered cofactor recruitment to PPARalpha as shown by coimmunoprecipitation from liver homogenates. In contrast, nearly all the respective free fatty acids elicited only weak conformational changes in PPARalpha and did not alter cofactor recruitment to PPARalpha. In summary, the CoA thioesters of very-long-chain and branched-chain fatty acids are much more potent PPARalpha ligands than the free acids, resulting in altered PPARalpha conformation and cofactor recruitment. Since these are hallmarks of ligand-activated nuclear receptors, this suggests that the CoA thioesters are the active forms of these PPARalpha ligands.