Members of species of the mulleri and hydei subgroups of the repleta group of Drosophila have duplicate Adh genes. The Adh regions of D. mojavensis, D. mulleri, and D. hydei contain three genes-a pseudogene, Adh-2, and Adh-1-arranged 5' to 3'. To understand the evolution of the triplicate Adh structure, we have cloned and sequenced the Adh locus of D. mettleri. This region consists of a 5' pseudogene and a 3' functional Adh gene. On the basis of the structure and nucleotide sequence comparisons of Adh genes of D. mettleri and other species, we propose that an initial duplication of the ancestral Adh gene generated two Adh genes arranged in tandem. The more 5' Adh gene became a pseudogene, while the more 3' gene remained functional through all the developmental stages. A second duplication of this 3' gene resulted in Adh regions with three genes-a pseudogene, Adh-2, and Adh-1.