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Liver carnitine palmitoyl transferase (L-CPT) I is a key regulatory enzyme of long-chain fatty acid (LCFA) oxidation that ensures the first step of LCFA import into the mitochondrial matrix. In rat hepatocytes, we showed previously that L-CPT I gene expression was induced by LCFAs as well as by fibrates. The aim of this study was to determine whether LCFA-induced L-CPT I gene expression was mediated by PPAR[alpha]. For this purpose, we constructed a PPAR[alpha]-dominant negative receptor to inhibit endogenous PPAR[alpha] signaling. Highly conserved hydrophobic and charged residues ([Leu.sup.459] and [Glu.sup.462]) in helix 12 of the ligand-binding domain were mutated to alanine. These mutations led to a total loss of transcriptional activity due to impaired coactivator recruitment. Furthermore, competition studies confirmed that the mutated PPAR[alpha] receptor abolished the wild-type PPAR[alpha] receptor action and thus acted as a powerful dominant negative receptor. When overexpressed in rat hepatoma cells (H4IIE) using a recombinant adenovirus, the mutated PPAR[alpha] receptor antagonized the clofibrate-induced L-CPT I gene expression, whereas it did not affect LCFA-induced L-CPT I. These results provide the first direct demonstration that LCFAs regulate L-CPT I transcription through a PPAR[alpha]-independent pathway, at least in hepatoma cells. KEY WORDS: * dominant negative PPAR[alpha] * recombinant adenovirus * carnitine palmitoyl transferase I * hepatoma cell * fatty acid-induced gene expression