Partial cDNA clones encoding human cytosolic aldehyde dehydrogenase (ALDH1) and mitochondrial aldehyde dehydrogenase (ALDH2) were isolated from a human liver cDNA library constructed in phage lambda gt11. The expression library was screened by using rabbit antibodies against ALDH1 and ALDH2. Positive clones thus obtained were subsequently screened with mixed synthetic oligonucleotides compatible with peptide sequences of ALDH1 and ALDH2. One of the positive clones for ALDH1 contained an insertion of 1.6 kilobase pairs (kbp). The insert encoded 340 amino acid residues and had a 3' noncoding region of 538 bp and a poly(A) segment. The amino acid sequence deduced from the cDNA sequence coincided with the reported amino acid sequence of human ALDH1 [Hempel, J., von Bahr-Lindström, H. & Jörnvall, H. (1984) Eur. J. Biochem. 141, 21-35], except that valine at position 161 in the previous amino acid sequence study was found to be isoleucine in the deduced sequence. Since the amino acid sequence of ALDH2 was unknown, 33 tryptic peptides of human ALDH2 were isolated and sequenced. Based on the amino acid sequence data thus obtained, a mixed oligonucleotide probe was prepared. Two positive clones, lambda ALDH2-21 and lambda ALDH2-36, contained the same insert of 1.2 kbp. Another clone, lambda ALDH2-22, contained an insert of 1.3 kbp. These two inserts contained an overlap region of 0.9 kbp. The combined cDNA contained a sequence that encodes 399 amino acid residues, a chain-termination codon, a 3' untranslated region of 403 bp, and a poly(A) segment. The deduced amino acid sequence was compatible with the amino acid sequences of the tryptic peptides. The degree of homology between human ALDH1 and ALDH2 is 66% for the coding regions of their cDNAs and 69% at the protein level. No significant homology was found in their 3' untranslated regions.