The nuclear protein LF-B1 (also referred to as HNF-1) is a transcription activator required for the expression of several liver-specific genes. LF-B1 has been purified to homogeneity from rat liver nuclear extracts. The sequence of the protein has been partially determined and, subsequently, overlapping cDNA clones containing the entire open reading frame of LF-B1 were isolated. The full-length cDNA encodes a 628 amino acid protein and directs the synthesis in vitro of a protein capable of binding DNA with the same specificity as LF-B1. The cDNA was recombined into a vaccinia virus vector and active LF-B1 was obtained from infected HeLa cells. Addition of the vaccinia recombinant protein to rat spleen extracts results in activation of transcription of an LF-B1-dependent promoter. The DNA binding domain of LF-B1 is located in the amino-terminal part of the protein and displays distant structural similarity to the homeobox domain. The distribution of LF-B1 mRNA is restricted to liver, which correlates with the tissue-specific expression of its target genes.