The human ALDH3 gene is constitutively expressed in stomach, lung, esophagus, and cornea, but hardly detectable in the normal liver. However, it is highly activated in the hepatocellular carcinoma tissues from approximately 50% of patients. The nuclear DNA binding factors exist in both ALDH3-positive cancerous liver and ALDH3-positive HepG2 cells, but not in ALDH3-negative Hep3B cells and normal liver tissues. South-western blot hybridization showed the existence of two nuclear-binding protein components, 35 and 14 kDa, in ALDH3-positive cancerous liver tissues. These two DNA binding proteins were not found in normal stomach tissues and stomach carcinoma KATO III cells. DNaseI footprint analysis identified two protective regions within the ALDH3 promoter. The first protected region has one putative CCAAT-box and one putative Sp1-site. The second protected region contains a putative HiNF-A binding sequence. These findings suggest that a high level of expression of ALDH3 in cancerous liver tissues resulted from the expression or activation of at least two nuclear proteins reacting to the ALDH3 promoter region.