We have cloned and determined the nucleotide sequence of a gene encoding alcohol dehydrogenase (Adh) from Triticum aestivum cv. Millewa. Southern analysis using cv. Chinese Spring nullisomic-tetrasomic and ditelosomic lines established that the cloned gene mapped to the long arm of chromosome 1A and does not correspond to any previously identified wheat Adh locus. Southern analysis also provided evidence for triplicate copies of this Adh gene on the homoeologous group 1 chromosomes, while Northern blots indicated that the homoeologous group 1 Adh genes, like several other plant Adh genes, are transcribed under anaerobic conditions. Sequence analysis indicates that the cloned gene has a structure similar to both monocot and dicot Adh genes with an open reading frame encoding a polypeptide of 379 amino acids. Sequences important for eucaryotic gene expression such as the TATA box, polyadenylation signal, and intron splice sites were found in the expected positions. The open reading frame is interrupted by 8 introns which are in identical positions with 8 of the 9 introns in maize and pea Adh genes, suggesting that during evolution there are processes occurring that result in the loss of introns. Sequence analysis also revealed that the cloned wheat Adh gene shared extensive homology with the barley Adh3 gene not only in the coding region but also in the noncoding regions. However, this homology is discontinuous as a result of a 1.8-kbp insertion (TLM), which is present in the cloned wheat Adh gene and absent in the barley Adh3 gene. Sequence analysis of this insertion reveals features characteristic of the short terminal inverted repeat class of eucaryotic transposable elements. We have no evidence for the transposition of the TLM element. However, Southern blots reveal multiple copies of sequences related to TLM in the wheat genome and in other closely related species, suggesting that transposition may once have played an important role in the evolution of the Gramineae family.