Outbreaks of unknown aetiology occur globally each year. They may be caused by the undetected release of a chemical, a novel pathogen or over exposure to ionizing radiation from a hidden source. They are usually detected by an increase in the incidence of unusual disease and therefore well-functioning surveillance systems are vital in this regard. Occasionally, the aetiological agent and/or the source or exposure pathway remain undetermined. However, thorough preparation and the implementation of a well-rehearsed and structured approach to outbreak investigation (including coordination of information, communication and analytical approaches) mean it is often possible to identify strong associations with particular exposures and thus limit the public health, economic and social effects of the outbreak. Drawing upon real-world case studies this chapter describes the investigation of outbreaks of unknown aetiology with a particular emphasis on those caused by chemicals. Some of the pitfalls and challenges that may be encountered are discussed.