Aldehyde reductase is involved in the reductive detoxification of reactive aldehydes that can modify cellular macromolecules. To analyze the mechanism of basal regulation of aldehyde reductase expression, we cloned the murine gene and adjacent regulatory region and compared it to the human gene. The mouse enzyme exhibits substrate specificity similar to that of the human enzyme, but with a 2-fold higher catalytic efficiency. In contrast to the mouse gene, the human aldehyde reductase gene has two alternatively spliced transcripts. A fragment of 57 bp is sufficient for 25% of human promoter activity and consists of two elements. The 3' element binds transcription factors of the Sp1 family. Gel-shift assays and chromatin immunoprecipitation as well as deletion/mutation analysis reveal that selenocysteine tRNA transcription activating factor (STAF) binds to the 5' element and drives constitutive expression of both mouse and human aldehyde reductase. Aldehyde reductase thus becomes the fourth protein-encoding gene regulated by STAF. The human, but not the mouse, promoter also binds C/EBP homologous protein (CHOP), which competes with STAF for the same binding site. Transfection of the human promoter into ethoxyquin-treated mouse 3T3 cells induces a 3.5-fold increase in promoter activity and a CHOP-C/EBP band appears on gel shifts performed with the 5' probe from the human aldehyde reductase promoter. Induction is attenuated in similar transfection studies of the mouse promoter. Mutation of the CHOP-binding site in the human promoter abolishes CHOP binding and significantly reduces ethoxyquin induction, suggesting that CHOP mediates stimulated expression in response to antioxidants in the human. This subtle difference in the human promoter suggests a further evolution of the promoter toward responsiveness to exogenous stress and/or toxins.