The genes that encode fructose 1,6-bisphosphate aldolase of Drosophila melanogaster have been isolated and characterized. These genes exist in a single copy 8-kilobase pair locus in the Drosophila genome which is located at cytogenetic position 97A-B. The nucleotide sequence and transcript mapping suggest that three overlapping protein isozyme genes may be encoded at this locus. These isozyme genes all share a single promoter, a 5'-untranslated first exon, and two other protein coding exons. The isozyme-specific carboxyl-terminal amino acids are encoded by one of three alternatively utilized fourth exons: 4A, 4B, or 4C by alternative splicing. The transcript containing exon 4C, whose sequence has been reported previously, is abundant throughout development and has a developmental profile similar to other glycolytic gene transcripts; however, it shows developmental specificity in the alternative use of two polyadenylation signals which result in a 2.4-kilobase and a 1.9-kilobase transcript. The transcript containing exon 4B is 1.6 kilobases in size and is most abundant during the larval stages and during the time of eclosion. The transcript containing exon 4A is in low abundance and found only during the adult stage. Sequence comparisons of the alternative fourth exons indicate that the duplication leading to the multiple exons is quite old and preceded the origin of the genus Drosophila.