Reactive oxygen intermediates (ROIs), such as hydrogen peroxide or superoxide, are an evolutionarily ancient threat to all organisms. Exposure of bacteria to ROIs initiates a genetic program that coordinates the production of novel proteins with protective functions. This genetic response is mediated by regulatory proteins that have the potential to initiate transcription of genes when the levels of the ROIs increase. In plant cells, a variety of viral pathogens increase hydrogen peroxide production, which is required to mount a defensive genetic response. It was suggested that in this case H2O2 is used as a secondary messenger and an immediate-early pathogen signal. In higher vertebrates, two transcription factors, nuclear factor kappa B and activator protein 1, were found to respond to ROIs. Both are well studied: they are induced by a great variety of seemingly unrelated conditions and serve important roles in immune, inflammatory, and other pathogen-related genetic responses. In this article we discuss how the ROI responsiveness of transcription factors can be experimentally studied and summarize evidence to suggest that ROIs have been conserved during evolution as messengers of a general pathogen response.