"Spot 14" protein appears rapidly in nuclei of hepatocytes exposed to glucose and thyroid hormone. Exposure of glucose- and T3-treated hepatocytes to a spot 14 antisense oligonucleotide inhibited induction of mRNAs encoding malic enzyme, ATP citrate-lyase, fatty acid synthase, liver-type pyruvate kinase, phosphoenolpyruvate carboxykinase, and type I deiodinase but not hydroxymethylglutaryl-CoA reductase, cytochrome c, and actin mRNAs. Induction of spot 14, ATP citrate-lyase, and fatty acid synthase polypeptides, but not propionyl-CoA carboxylase and mitochondrial pyruvate carboxylase, was inhibited. Antisense treatment of hepatocytes transfected with a reporter controlled by a glucose- and T3-inducible fragment of the pyruvate kinase gene promoter inhibited reporter activity, as did cotransfection of the reporter and a spot 14 antisense plasmid. Spot 14 protein acts in the induction of mRNAs coding for key lipogenic (malic enzyme, ATP citrate-lyase, fatty acid synthase), glycolytic (pyruvate kinase), and gluconeogenic enzymes (phosphoenolpyruvate carboxykinase), as well as the diet-responsive type I deiodinase, but not those involved in mitochondrial respiration (cytochrome c) or cholesterol synthesis (hydroxymethylglutaryl-CoA reductase). Transfection experiments indicated that these effects are mediated at the transcriptional level. The protein functions in the activation of genes involved in metabolic switching between the fasted and fed states in liver.