An actin filament binding protein cortactin was initially identified as a major phosphotyrosine-containing protein in v-Src-transformed chicken embryo fibroblast cells. The mouse, human, and Drosophila homologs were independently identified as a signaling molecule involved in a mitogenic response, as a product of a putative oncogene EMS1, and as a molecule interacting with a scaffolding protein ZO-1, respectively. In this report, we describe the cloning of the Drosophila cortactin gene, which consists of four exons and three introns, covering 3 kilobases in length. All exon-intron junctions are well matched with the GT/AG consensus sequence. S1 nuclease mapping revealed one major and several minor transcription start sites. The cytological location of the Drosophila cortactin gene is between chromosome segments 93B3 and 93B7.