We have isolated and characterized several overlapping clones from two human genomic libraries constructed in cosmid and bacteriophage vectors. They span about 80 kbp and include the entire human cytosolic aldehyde dehydrogenase (ALDH1) gene. Restriction endonuclease mapping, Southern blotting with cDNA and specific oligonucleotide probes, and DNA sequencing were performed to analyze the cloned genomic DNA. The ALDH1 gene is about 53 kbp long and is divided into 13 exons which encode 501 amino acid residues. Primer extension results defined the transcription initiation site to 53 bp upstream from the A of the initiation codon ATG. The promoter region of the gene contains an ATA box and a CCAAT box, which are located 32 and 74 bp upstream, respectively, from the transcription initiation site. The possible functional domains of the protein encoded by exons are discussed. A similar intron-exon organization between the genes of cytosolic ALDH1 and its mitochondrial ALDH2 isozyme in which both enzymes are encoded by 13 exons and 9 of the 12 introns interrupt the coding sequence at homologous positions was observed. This is consistent with the model that the two isozyme genes evolved after the duplication of a common ancestor gene.