Here we examine the molecular basis for the known preferential expression of rabbit aldehyde dehydrogenase class 1 (ALDH1A1) in the cornea. The rabbit Aldh1a1 promoter-firefly luciferase reporter transgene (-3519 to +43) was expressed preferentially in corneal cells in transfection tests and in transgenic mice, with an expression pattern resembling that of rabbit Aldh1a1. The 5' flanking region of the rabbit Aldh1a1 gene resembled that in the human gene (60.2%) more closely than that in the mouse (46%) or rat (51.5%) genes. We detected three xenobiotic response elements (XREs) and one E-box consensus sequence in the rabbit Aldh1a1 upstream region; these elements are prevalent in other highly expressed corneal genes and can mediate stimulation by dioxin and repression by CoCl(2), which simulates hypoxia. The rabbit Aldh1a1 promoter was stimulated fourfold by dioxin in human hepatoma cells and repressed threefold by CoCl(2) treatment in rabbit corneal stromal and epithelial cells. Cotransfection, mutagenesis, and gel retardation experiments implicated the hypoxia-inducible factor 3alpha/aryl hydrocarbon nuclear translocator heterodimer for Aldh1a1 promoter activation via the XREs and stimulated by retinoic acid protein 13 for promoter repression via the E-box. These experiments suggest that XREs, E-boxes, and PAS domain/basic helix-loop-helix transcription factors (bHLH-PAS) contribute to preferential rabbit Aldh1a1 promoter activity in the cornea, implicating hypoxia-related pathways.