The rate-limiting step in 20:4(n-6) and 22:6(n-3) synthesis is the desaturation of 18:2(n-6) and 18:3(n-3) by Delta-6 desaturase. In this report, we demonstrate that n-6 and n-3 PUFAs suppressed the hepatic expression of rodent Delta-6 desaturase by inhibiting the rate of Delta-6 desaturase gene transcription. In contrast, consumption of the peroxisome proliferator-activated receptor (PPAR)alpha activator WY 14,643 significantly enhanced the transcription of hepatic Delta-6 desaturase by more than 500%. Transfection reporter assays with HepG2 cells revealed that the PUFA response region for the human Delta-6 desaturase gene involved the proximal promoter region of -283/+1 human Delta-6 desaturase gene, while the WY 14,643 response element (RE) was identified as an imperfect direct repeat (DR-1) located at -385/-373. The WY 14,643 induction of the human Delta-6 desaturase promoter activity was dependent upon the expression of PPARalpha. Electrophoretic mobility shift assays revealed that nuclear proteins extracted from HepG2 cells expressing PPARalpha specifically interacted with the -385/-373 DR-1 sequence of the human Delta-6 desaturase gene. The interaction was eliminated by the unlabeled PPARalpha RE of the rat acyl-CoA oxidase gene, and the protein-DNA complex was super-shifted by treatment with anti-PPARalpha. The -385/-373 sequence also interacted with a mixture of in vitro translated PPARalpha-retinoic acid receptor X (RXR)alpha, but by themselves neither PPARalpha nor RXRalpha could bind to the Delta-6 desaturase DR-1. These data indicate that the 5'-flanking region of the human Delta-6 desaturase gene contains a DR-1 that functions in the regulation of human Delta-6 desaturase gene transcription, and thereby plays a role in the synthesis of 20- and 22-carbon polyenoic fatty acids.