The gene that codes for Drosophila alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase EC 18.104.22.168) was identified in a bacteriophage lambda library of genomic Drosophila DNA by using ADH cDNA cloned DNA as a probe. The DNA sequence of the protein encoding region was shown to be in agreement with the amino acid sequence of the ADH. Two intervening DNA sequences (introns) were identified within the protein encoding region: one was 65 nucleotides and located between the codons for amino acid residues 32 and 33, and one was 70 nucleotides and located between the codons for amino acid residues 167 and 168. Both contained the 5' G-T and 3' A-G dinucleotides characteristic of intron boundaries of eukaryotic genes. On the basis of secondary structure predictions, the first 140 amino acid residues of Drosophila ADH are in an alternating beta-sheet/alpha-helix arrangement which is characteristic of the coenzyme binding domain of dehydrogenases. The smaller of the two introns interrupts the domain predicted to bind the adenine portion of the coenzyme.