We report the isolation of the genomic sequence that encodes the enzyme triose phosphate isomerase of Drosophila melanogaster. There is a single copy of the Tpi sequence in the genome of Drosophila, as judged by Southern blots and in situ hybridization to salivary gland chromosomes. The sequence of 3414 nucleotides from the Tpi region was determined. The gene has an intron in the 5' untranslated region of the transcript and a second intron in the coding region at an evolutionarily conserved position. Transcripts initiate at a single site which does not have a TATA box in the usual position. Northern blot analysis of RNA prepared from different developmental stages revealed that Tpi mRNA is present in substantial amounts in oocytes, declines in abundance in early embryos, and begins to increase during mid-embryogenesis. Transcript abundance follows a pattern typical of enzymes involved in intermediate metabolism. A peak is found during third instar followed by a decline during pupal stages and then a second rise near the time of eclosion.