Heme oxygenase-1 is the rate-limiting enzyme for the degradation of the prooxidant heme. Previously, we showed that an E-box within the HO-1 promoter is crucial for the regulation of HO-1 expression in primary hepatocytes. Further to investigate the importance of this E-box, we determined the regulatory capacity of the E-box-binding factor USF-2 in primary cells in comparison with transformed cell lines. We found that HO-1 expression was inhibited by USF-2 in primary cells, whereas it was induced in tumor cell lines. Mutation of either the E-box or the AP-1 site within the HO-1 promoter only partially affected the USF-dependent regulation. However, this regulation was dramatically reduced in tumor cells and completely abolished in primary cells transfected with an HO-1 promoter construct containing mutations in both the E-box and the AP-1 site, suggesting that AP-1 factors and USF-2 may act in a cooperative manner. Indeed, protein-protein interaction studies revealed that USF proteins interacted with Fra-1. Further, the USF-dependent HO-1 promoter activity was not detectable with an USF-2 mutant lacking residues of the USF-specific region (USR) or the transactivation domain encoded by exon 4. Together, these data suggest that USF-2 has opposite regulatory roles for HO-1 gene expression in primary cells and tumor cell lines.