Human alcohol dehydrogenase (ADH) is encoded by at least five genes that fall into three classes. The class I ADH genes encode the three closely related α, β, and γ polypeptides. Molecular genetic analysis of class I ADH genes has been initiated by isolating a cDNA clone from a human adult liver cDNA library. A synthetic oligonucleotide mixture encoding a portion of the β subunit of ADH was used as an in situ hybridization probe for the cDNA library. One positively hybridizing clone, pADH12, which contained an 1100-base-pair cDNA insert, was subjected to DNA sequence analysis. The sequence indicated that the cDNA encoded information for the carboxyl-terminal 91 amino acids of a class I ADH and a 3' untranslated region of 593 nucleotides. Comparisons with the carboxyl terminus of the human ADH β subunit indicated that the cDNA encoded the β polypeptide. This probe may facilitate genetic studies of various human alcohol-related syndromes, as well as enable basic molecular studies on human ADH gene expression.