Several missense mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor (PPAR)γ have been described in subjects with dominantly inherited severe insulin resistance associated with partial lipodystrophy, hypertension, and dyslipidemia. These mutant receptors behave as dominant-negative inhibitors of PPARγ signaling when studied in transfected cells. The extent to which such dominant-negative effects extend to signaling through other coexpressed PPAR isoforms has not been evaluated. To examine these issues further, we have created a PPARα mutant harboring twin substitutions, Leu459Ala and Glu462Ala, within the ligand binding domain (PPARα_mut), examined its signaling properties, and compared the effects of dominant-negative PPARα and PPARγ mutants on basal and ligand-induced gene transcription in adipocytes and hepatocytes. PPARα_mut was transcriptionally inactive, repressed basal activity from a PPAR response element-containing promoter, inhibited the coactivator function of cotransfected PPAR-γ coactivator 1α, and strongly inhibited the transcriptional response to cotransfected wild-type receptor. In contrast to PPARγ, wild-type PPARα failed to recruit the transcriptional corepressors NCoR and SMRT. However, PPARα_mut avidly recruited these corepressors in a ligand-dissociable manner. In hepatocytes and adipocytes, both PPARα_mut and the corresponding PPARγ mutant were capable of inhibiting the expression of genes primarily regulated by PPARα, -γ, or -δ ligands, albeit with some differences in potency. Thus, dominant-negative forms of PPARα and PPARγ are capable of interfering with PPAR signaling in a manner that is not wholly restricted to their cognate target genes. These findings may have implications for the pathogenesis of human syndromes resulting from mutations in this family of transcription factors.