Rat aflatoxin B(1) aldehyde reductase (called AFAR1 or AKR7A1) is a member of the aldo-keto reductase 7 family, which metabolizes the environmental carcinogen aflatoxin B(1). The expression of this enzyme is markedly increased in rat liver by cancer chemopreventive agents, many of which are believed to regulate gene expression through the antioxidant response element (ARE). In order to understand how this gene is regulated, two overlapping genomic clones have been isolated that contain most of the coding region for the enzyme; together they encompass 14.1 kb of DNA. Characterization of these clones has shown that rat AFAR1 is approximately 8 kb long and comprises seven exons and six introns. The seven exons are between 97 and 380 bp in size. The introns range in size from 194 bp to approximately 2.9 kb. Fluorescent in situ hybridization localized AFAR1 to rat chromosome 5q36.5, a region that is syntenic with human chromosome 1p35-1p36.1 where AKR7A2 resides. The transcriptional start site (TSS) was determined, using 5'-rapid amplification of cDNA ends, to be an A nucleotide 73 bp upstream from the ATG initiation codon. The 5'-flanking region of AFAR1 was isolated by polymerase chain reaction-based genome walking, and resulted in the isolation of approximately 900 bp of genomic DNA upstream from the TSS. Use of a gene expression reporter assay demonstrated that this cloned 5'-flanking region of AFAR1 could support transcription in the rat liver 34 (RL34) epithelial cell line. Within this upstream region of the promoter, a substantial number of sequences were found that are closely similar, but not identical, to the 'core' ARE consensus sequence. Between nucleotides -810 and -106 bp from the TSS 16 ARE-related sequences were identified. Four of these putative enhancers lay between -389 and -355 bp, and the motif 5'-GAGTGAG-3' was repeated three times within the 35 bp region.