Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a central regulator of lipid metabolism. Fibrate drugs act on PPARalpha to modulate dyslipidemias. A natural variant (V227A) affecting the PPARalpha hinge region was associated with perturbations in blood lipid levels in Asian populations. In this study, we investigated the functional significance of the V227A substitution. The variant significantly attenuated PPARalpha-mediated transactivation of the cytochrome P450 4A6 and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2) genes in the presence of fibrate ligands. Screening of a panel of PPARalpha coregulators revealed that V227A enhanced recruitment of the nuclear corepressor NCoR. Transactivation activity of V227A could be restored by silencing NCoR or by inhibition of its histone deacetylase activity. Deletion studies indicated that PPARalpha interacted with NCoR receptor-interacting domain 1 (ID1) but not ID2 or ID3. These interactions were dependent on the intact consensus nonapeptide nuclear receptor interaction motif in NCoR ID1 and were enhanced by the adjacent 24 N-terminal residues. Novel corepressor interaction determinants involving PPARalpha helices 1 and 2 were identified. In hepatic cells, the V227A substitution stabilized PPARalpha/NCoR interactions and caused defective release of NCoR in the presence of agonists on the HMGCS2 promoter. These results provide the first indication that defective function of a natural PPARalpha variant was due, at least partially, to increased corepressor binding. Our data suggest that the PPARalpha/NCoR interaction is physiologically relevant and can produce a discernable phenotype when the magnitude of the interaction is altered by a naturally occurring variation.