Glucose-6-phosphatase confers on gluconeogenic tissues the capacity to release endogenous glucose in blood. The expression of its gene is modulated by nutritional mechanisms dependent on dietary fatty acids, with specific inhibitory effects of polyunsaturated fatty acids (PUFA). The presence of consensus binding sites of hepatocyte nuclear factor 4 (HNF4) in the -1640/+60 bp region of the rat glucose-6-phosphatase gene has led us to consider the hypothesis that HNF4 alpha could be involved in the regulation of glucose-6-phosphatase gene transcription by long chain fatty acid (LCFA). Our results have shown that the glucose-6-phosphatase promoter activity is specifically inhibited in the presence of PUFA in HepG2 hepatoma cells, whereas saturated LCFA have no effect. In HeLa cells, the glucose-6-phosphatase promoter activity is induced by the co-expression of HNF4 alpha or HNF1 alpha. PUFA repress the promoter activity only in HNF4 alpha-cotransfected HeLa cells, whereas they have no effects on the promoter activity in HNF1 alpha-cotransfected HeLa cells. From gel shift mobility assays, deletion, and mutagenesis experiments, two specific binding sequences have been identified that appear able to account for both transactivation by HNF4 alpha and regulation by LCFA in cells. The binding of HNF4 alpha to its cognate sites is specifically inhibited by polyunsaturated fatty acyl coenzyme A in vitro. These data strongly suggest that the mechanism by which PUFA suppress the glucose-6-phosphatase gene transcription involves an inhibition of the binding of HNF4 alpha to its cognate sites in the presence of polyunsaturated fatty acyl-CoA thioesters.