Single-copy DNA was isolated from Drosophila melanogaster and hybridized with total genomic DNA of D. melanogaster, D. mauritiana, D. simulans, D. pseudoobscura, D. willistoni, D. hydei and D. virilis. The duplexes were thermally eluted from hydroxyapatite and the data used to assess the relatedness of each species to D. melanogaster. The general pattern of relatedness was similar to that predicted by morphological methods but with some notable exceptions. The rate of nucleotide substitution was estimated to be greater than 0.66% of bases per million years. An unexpected, rapidly evolving component of D. melanogaster single-copy DNA was identified. The relatedness of these species was also studied with respect to the gene coding for alcohol dehydrogenase (ADH). The ADH gene, previously cloned from D. melanogaster (Goldberg 1980), was hybridized with Southern blots of genomic digests of the seven species. The intensity and position of the hybridizing bands suggest the amount of divergence of the gene. Divergence was quantitated by reassociation of a fragment of the cloned ADH gene with total DNA of the seven drosophilids and thermal elution of the resultant duplexes from hydroxyapatite. The ADH gene was isolated from genomic clone libraries of D. melanogaster, D. simulans and D. mauritiana and further studied by comparison of position of restriction sites. Species relationships deduced from comparison of total single-copy DNA and the ADH gene were consistent, demonstrating that a single gene can reflect divergence of the entire genome.