Hepatic cell‐specific expression of the human apolipoprotein B (apoB) gene is controlled by at least four cis‐acting elements located between positions −128 and +122 [Chuang, S. S., & Das, H. K. (1996) Biochem. Biophys. Res. Commun.220, 553–562]. A negative cis‐acting element (+20 to +40) is located in the first nontranslated exon of the human apoB gene, and apoB regulatory factor‐3 (BRF‐3) interacts with this. In this paper, we report the purification and characterization of BRF‐3 from rat liver nuclear extracts. BRF‐3 has been purified to apparent homogeneity by DEAE–cellulose, heparin–agarose, and DNA‐specific affinity chromatography. Purified BRF‐3 produced two polypeptide bands with apparent molecular masses of 70 kDa and 67 kDa in SDS/PAGE as detected by silver staining. Both 70‐kDa and 67‐kDa proteins have been found to hybridize specifically with labeled double‐stranded oligonucleotide containing BRF‐3 binding site in a South‐Western blot. Double‐stranded oligonucleotide containing mutations in the BRF‐3 binding site was found to abolish DNA binding by these two proteins. Amino acid sequences of tryptic peptides derived from affinity purified 70‐kDa and 67‐kDa rat BRF‐3 proteins were found to have 100% sequence homologies with DNA topoisomerase I. These data suggest that the 70‐kDa and 67‐kDa forms of BRF‐3 are derived by proteolytic cleavage of topoisomerase I, and therefore, topoisomerase I may play an important role in transcriptional regulation of apoB.