The differentiation-specific element (DSE) is a cis-acting transcriptional element located at nucleotide--1000 in the 5'-flanking promoter of the angiotensinogen gene. It is required for the irreversible and sustained increase in transcription of the angiotensinogen gene that occurs during differentiation of 3T3-L1 adipoblasts into adipocytes induced by a 3-day hormonal pulse. We report here the cloning of 3T3-L1 adipocyte cDNA encoding a 150 kilodalton protein designated Differentiation Specific Element Binding Protein (DSEB) that exhibits sequence-specific binding to a DSE oligonucleotide. Two DSEB mRNAs (3.6 and 4.2 kilobases) are observed in adipose, brain, kidney, testis, liver, and lung. Both DSEB mRNA and protein are induced during, and remain elevated after, 3T3-L1 cell adipogenesis. Analysis of adipoblasts by immunocytochemistry with an antiserum directed to bacterial expressed DSEB reveals that DSEB is localized to the nucleus and is induced during differentiation. DNA-binding assays show that binding is specific and exhibits high affinity and specificity for the DSE. Deletional analyses of bacterial expressed recombinant DSEB identifies a DNA-binding domain of 120 amino acids that contains two predicted helical regions. A sequence of 72 amino acids within the DNA-binding domain of DSEB is 60% identical to domains found in the sequences of several bacterial ligases. Further, DSEB is homologous to several proteins reported recently that are proposed to be a component(s) of the DNA replication-C complex raising the possibility that DSEB may be both a transcription factor and a DNA-replication factor.