To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to −176) that contains a single E‐box and an adjacent nuclear factor 1 (NF1)‐like DNA binding site. The E‐box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA–protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E‐box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF‐1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1‐like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:78–86, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20063