HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds to sites required for the transcription of the genes for transthyretin (TTR), the carrier protein in the serum for vitamin A and thyroid hormone, and for apolipoprotein CIII (apoCIII), a major constituent of chylomicrons and very low-density lipoproteins (VLDL). Synthetic oligonucleotides derived from amino acid sequence of affinity-purified HNF-4 protein (54 kD) were used in the polymerase chain reaction (PCR) to isolate a cDNA clone encoding the protein. HNF-4 is a member of the steroid hormone receptor superfamily with an unusual amino acid in the conserved" knuckle" of the first zinc finger (DGCKG). Studies with in vitro-translated HNF-4 protein show that it binds to its recognition site as a dimer, and cotransfection assays indicate that it activates transcription in a sequence-specific fashion in nonhepatic (HeLa) cells. Northern blot analysis reveals that HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues. DNA-binding and antisera reactivity data suggest that HNF-4 could be identical to liver factor A1 (LF-A1), a DNA-binding activity implicated in the regulation of transcription of the alpha 1-antitrypsin, apolipoprotein A1, and pyruvate kinase genes. The similarity between HNF-4 and other ligand-dependent transcription factors raises the possibility that HNF-4 and the genes it regulates respond to an as yet unidentified ligand.