The alcohol dehydrogenase (Adh) gene has been isolated from Drosophila simulans and D. mauritiana by screening lambda clone libraries of each with a previously cloned Adh gene from D. melanogaster. The isolated lambda clones were subcloned and partially sequenced to determine the relatedness of these species and to examine details of evolutionary change in the structure of the Adh gene. We report the sequence of the first 704 nucleotides of each gene as well as 127 bases in the 5' untranslated region. When these sequences are compared, D. melanogaster differs from D. simulans and D. mauritiana by 2.8% and 3.1%, respectively. D. simulans and D. mauritiana differ by only 1.8%, implying that they are more closely related to each other than either is to D. melanogaster. This is consistent with phylogenetic relationships established by a variety of genetic, biochemical, and morphological means and illustrates that DNA sequencing of a single gene may be used to assess the evolutionary relationships of species.