Amatoxins are a group of natural toxins which occur in the death cap mushroom (Amanita phalloides). They work by inhibiting RNA polymerase II, which results in apoptosis. RNA-polymerase II inhibition is a novel mechanism of action in cancer therapy and offers the possibility of breaking through drug resistance or destroying dormant tumour cells, which could produce major clinical advances. Amanitin, as the most potent member of this toxin family, has been made accessible for cancer therapy by developing it as a payload for antibody–drug conjugates (ADCs). This chapter describes the discovery and chemistry of the amatoxins, and the development of the amanitin-ADC technology.