Chagas disease and African sleeping sickness are trypanosomal infections that represent important public health problems in Latin America and Africa, respectively. The restriction of these diseases to the poorer parts of the world has meant that they have been largely neglected and limited progress has been made in their treatment. The nitroheterocyclic prodrugs nifurtimox and benznidazole, in use against Chagas disease for >40 years, remain the only agents available for this infection. In the case of African sleeping sickness, nifurtimox has recently been added to the arsenal of medicines, with the nitroheterocycle fexinidazole currently under evaluation. For a long time, the cytotoxic mechanism of these drugs was poorly understood: nifurtimox was thought to act via production of superoxide anions and nitro radicals, while the mode of benznidazole action was more obscure. The trypanocidal activity of nitroheterocyclic drugs is now known to depend on a parasite type I nitroreductase (NTR). This enzyme is absent from mammalian cells, a difference that forms the basis for the drug selectivity. The role of this enzyme in drug activation has been genetically and biochemically validated. It catalyses the 2-electron reduction of nitroheterocyclic compounds within the parasite, producing toxic metabolites without significant generation of superoxide. Recognition that this enzyme is responsible for activation of nitroheterocyclic prodrugs has allowed screening for compounds that preferentially target the parasite. This approach has led to the identification of two new classes of anti-trypanosomal agents, nitrobenzylphosphoramide mustards and aziridinyl nitrobenzamides, and promises to yield new, safer, more effective drugs.