The nuclear receptor (NR) peroxisome proliferator-activated receptor-α (PPARα) mediates the effects of several hypolipidemic drugs, endogenous fatty acids, and peroxisome proliferators. Despite belonging to a class of NR not known to interact with cytosolic chaperone complexes, we have recently shown that PPARα interacts with heat shock protein 90 (Hsp90), although the biological consequence of this association was unknown. In the present study, PPARα directly associated with Hsp90 in vitro to a much greater extent than either PPARβ or PPARγ. This interaction is similar to other NR-Hsp90 complexes with association occurring between the middle of Hsp90 and the hinge (D) and ligand binding domain (EF) of PPARα. Using several different approaches to disrupt Hsp90 complexes within the cell, we demonstrate that Hsp90 is a repressor of both PPARα and PPARβ activity. Treatment with geldanamycin (GA) increased the activity of PPARα and in the presence of ligand in transient transfection assays. PPARα-response element (PPRE)-reporter assays in a stable cell line treated with GA resulted in enhanced expression of a known target gene, acyl-CoA oxidase. Similarly, overexpression of the tetratricopeptide repeat (TPR) of protein phosphatase 5 (PP5) increased PPARα or PPARβ activity in a PPRE-reporter assay and decreased the interaction between PPARα or PPARβ and Hsp90 in a mammalian two-hybrid assay. Finally, cotransfection with the C-terminal hsp-interacting protein (CHIP) construct, a TPR-containing ubiquitin ligase that interacts with hsp90, increased PPARα's and decreased PPARβ's ability to regulate PPRE-reporter activity upon ligand activation. All three methods to disrupt Hsp90 function (GA, PP5-TPR, CHIP) resulted in an alteration in PPARα or PPARβ activity to a much greater extent than PPARγ. While FKBP52 had no effect on PPARα activity, p23 greatly enhanced constitutive and Wy14 643 induced PPRE-reporter activity. Thus, we describe the chaperone complex as being a regulator of PPARα and PPARβ activity and have identified a novel, subtype-specific, inhibitory role for Hsp90.