The gene from Drosophila melanogaster that encodes phosphofructokinase has been isolated and its structure and expression characterized. Southern blots of genomic DNA, comparison of multiple clones, and in situ hybridization to salivary gland chromosomes indicate that the Drosophila genome contains a single copy of the gene which encodes phosphofructokinase (PFK) that is located at cytogenetic position 46D/E on the polytene map. Analysis of the gene structure reveals a single coding region that spans 6.5 kilobase pairs and is composed of eight exons and seven introns. Three developmentally regulated transcripts are generated through the use of alternative polyadenylation sites. Transcription initiation occurs at a single site. The pattern of transcript accumulation during development is similar to other genes of Drosophila which encode enzymes of the glycolytic pathway. Deficiency analysis of the 46D/E region has identified a lethal complementation group associated with reduced PFK activity, thereby establishing the vital role of PFK function in Drosophila.