The mouse cytosolic aldehyde dehydrogenase ALDH3A1 (encoded by the Aldh3a1 gene) has previously been shown in cell culture to be markedly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), downregulated by the metabolism of functional CYP1A1/1A2 enzymes, and upregulated by a gene on Chr 7 that leads to endogenous oxidative stress. In order to study the regulation of Aldh3a1 gene expression, we isolated two overlapping genomic sequences from a B6/CBA mouse genomic library that included the entire Aldh3a1 gene, along with considerable 5' and 3' flanking sequences. The Aldh3a1 gene was shown to span approximately 10 kb and comprise 11 exons including a noncoding first exon. The sequence of 3.18 kb upstream of exon 1 reveals numerous consensus transcription factor-binding sites, some of which were shown to be important in the positive and negative control of Aldh3a1 gene expression; these include seven aromatic hydrocarbon response elements (AHREs), an electrophile response element (EPRE), and AP-1, C/EBP beta, c/EBP alpha, NF-kappaB, Sp1, and NF-1 putative binding sites. Deletion fusion constructs containing regions of the Aldh3a1 gene 5' flanking sequence, ligated to chloramphenicol experiments suggested that the 5' flanking region of the gene contains a strong promoter, at least four functional AHREs appear to act cooperatively in causing dioxin-mediated upregulation, and a putative negative regulatory element (NRE) controls basal gene expression independent of dioxin inducibility. The dioxin-mediated upregulation of Aldh3a1 expression in mouse hepatoma Hepa-1c1c7 cell cultures was shown to depend exclusively on the aromatic hydrocarbon receptor. acetyltransferase (CAT) or luciferase (LUC) reporter genes, were studied. Transient transfection experiments suggested that the 5' flanking region of the gene contains a strong promoter, at least four functional AHREs appear to act cooperatively in causing dioxin-mediated upregulation, and a putative negative regulatory element (NRE) controls basal gene expression independent of dioxin inducibility. The dioxin-mediated upregulation of Aldh3a1 expression in mouse hepatoma Hepa-1c1c7 cell cultures was shown to depend exclusively on the aromatic hydrocarbon receptor.