Class IV alcohol dehydrogenase (ADH) has been shown to function in vitro as a retinol dehydrogenase catalyzing the synthesis of retinoic acid, a pleiotropic gene regulator. To enable genetic studies on the function of this enzyme and regulation of its gene, we have screened a genomic library and isolated the mouse class IV ADH gene (Adh3). The complete mouse class IV ADH coding region was found in nine exons spanning a 14-kb region. Primer extension analysis was used to map the transcription initiation site to a position lying 30 bp upstream of the ATG translation start codon. Nucleotide sequence analysis of the promoter region indicated an absence of both TATA-box and GC-box sequences; this distinguishes it from the promoters for class I, II, and III ADH genes. Sequence comparison of the mouse and human class IV ADH promoters indicated that they share a conserved region located 125-145 bp upstream of the coding region containing adjacent sequences matching the consensus binding sites for transcription factors AP-1 and C/EBP.