AbstractAfrican sleeping sickness, Chagas disease, and leishmaniasis are life-threatening diseases that together affect millions of people around the world and are caused by different members of the protozoan familyTrypanosomatidae. The most studied member of the family isTrypanosoma brucei, which is spread by tsetse flies and causes African sleeping sickness. Nucleotide metabolism inT. bruceiand other trypanosomatids is significantly different from that of mammals and was recognized as a target for chemotherapy already in the 1970–1980s. A more thorough investigation of the nucleotide metabolism in recent years has paved the way for identifying nucleoside analogues that can cureT. bruceibrain infections in animal models. Specific features ofT. bruceinucleotide metabolism include the lack ofde novopurine biosynthesis, the presence of very efficient purine transporters, the lack of salvage pathways for CTP synthesis, unique enzyme localizations, and a recently discovered novel pathway for dTTP synthesis. This review describes the nucleotide metabolism ofT. brucei, highlights differences and similarities to other trypanosomatids, and discusses how to exploit the parasite-specific features for drug development.