This Review describes how microbial or self DNA that enters the cytoplasm can be detected by various mechanisms and triggers a range of cellular responses. These include the induction of antiviral innate immune responses and inflammasome-dependent caspase-1 activation and pyroptotic cell death. The recognition of nucleic acids is one strategy by which cells can detect infectious agents. As life is ultimately determined by the existence of nucleic acids, this defence strategy has evolved in many different organisms and operates effectively in many different cell types. Here, we review the recent progress in our understanding of the molecular mechanisms by which DNA activates cells to induce inflammation and antimicrobial immunity. DNA can be detected in different cellular compartments and can induce a range of cellular responses, such as an antiviral response and pyroptotic cell death together with the maturation and release of active interleukin-1β.