PPARα is a nuclear receptor that controls lipid and glucose metabolism and exerts antiinflammatory activities. The factors regulating human PPARα (hPPARα) gene expression remain largely unexplored. To study the mechanisms controlling hPPARα expression, the hPPARα gene promoter was identified and characterized. First, an alternatively spliced exon within the 5′-untranslated region of the hPPARα gene was identified by RT-PCR. Next, the transcription start site was mapped and the hPPARα gene promoter was cloned and functionally analyzed. Because PPARα levels are elevated in tissues expressing the hepatocyte nuclear factor-4 (HNF4), such as liver, the regulation of hPPARα by HNF4 was examined. Transient transfections in HepG2 and Cos cells showed that HNF4 enhances hPPARα promoter activity. 5′-Deletion and mutation analysis of the hPPARα promoter identified a regulatory element (RE) consisting of a degenerate hexamer repeat with a single nucleotide spacer (direct repeat 1), termed αHNF4-RE. Gel shift assays demonstrated that HNF4 binds to this αHNF4-RE. Furthermore, HNF4 increased the activity of a heterologous promoter driven by two copies of the αHNF4-RE. The nuclear receptor COUP-TFII also bound this site and down-regulated basal as well as HNF4-induced hPPARα promoter activity. Finally, PPARα was shown to bind the αHNF4-RE, leading to an induction of PPARα expression in hepatocytes. In summary, the organization of the 5′-flanking and untranslated region of the hPPARα gene was characterized and the hPPARα promoter region has been identified. Furthermore, these data demonstrate that the hPPARα gene is regulated by nuclear receptors, such as HNF-4, COUP-TFII, and PPARα.