Eukaryotic cells sequester their DNA in the nucleus and organelles, leaving cytosol DNA-free. The presence of DNA in the cytosol indicates a danger, such as infection, activity of retrotransposons or DNA damage. We hypothesized that defence against cytosolic DNA is a feature of all eukaryotic cells, and fundamental to maintenance of genome integrity as well as recognition of infection. In mammals cytosolic DNA is recognized by AIM2, a member of the HIN-200 family, leading to formation of a multimolecular inflammasome complex, that in turn induces a rapid lytic death in macrophages, termed pyroptosis. There is no data about such a system for invertebrates, and AIM2 itself is a mammalian-restricted protein. Moreover there is no indication of active necrotic cell death in insects. To investigate whether an analogous system operates in Drosophila, cells were transfected with different types of DNA via electroporation and viability was assessed either by MTT assay or by exclusion of membrane-impermeable DNA stains. We have found that both primary hemocytes, and the Drosophila S2 macrophage-like cell line are sensitive to cytosolic DNA. Results showed that DNA from various sources, but not synthetic double stranded RNA caused rapid cell death. Unlike mammalian DNA-dependent cell death, denatured DNA was also toxic. Experiments with synthetic DNA showed that the response was sequence-specific. The mode of cell death was investigated using flow cytometry. Rapid loss of membrane integrity, lack of a population of cells with sub-G0/G1 levels of DNA, and insensitivity to the pancaspase inhibitor z-VAD-fmk showed that cell death was not apoptotic. We conclude that sensing of cytosolic DNA in fruitfly induced a novel lytic pathway of cell death. Elucidation of the molecular basis for this DNA recognition will allow determination of whether it plays a role in defence against infection or in guarding genome integrity.