Topoisomerases are essential for orderly nucleic acid metabolism and for cell survival, and are proven targets for clinically useful antimicrobial and anticancer drugs. Interest in the topologically intricate mitochondrial DNA (kinetoplast, or kDNA) of Trypanosoma brucei brucei and related kinetoplastid protozoan parasites has led to many reports of type II topoisomerases that participate in kDNA metabolism (we term the T. b. brucei gene TbTOP2mt). We have now identified and characterized two new genes for type II topoisomerases in T. b. brucei, termed TbTOP2a and TbTOP2ß. Phylogenetically they share a common node with other nuclear topoisomerases, clearly distinct from a clade which includes the previously reported kinetoplastid genes, all of which are homologs of TbTOP2mt. Southern blot analysis reveals the new genes are single copy and positioned ~1.7 kb apart. Cognate mRNAs are expressed in African trypanosomes but only a single message is detected in Leishmania or Crithidia. TbTOP2a encodes an ATP-dependent topoisomerase which appears as a single ~170 kDa band on immunoblots and which localizes to the nucleus; RNA interference leads to pleomorphic nuclear (but not kDNA) abnormalities and early growth arrest. The role of TbTOP2ß is unclear. Though transcribed in trypanosomes, TbTOP2ß is not detected by ß-specific antiserum and RNAi silencing results in no obvious phenotype. These studies indicate that African trypanosomes and related kinetoplastid human pathogens are unusual in having independent topoisomerase II genes to service their nuclear and mitochondrial genomes, and they highlight TbTOP2a as a promising target for the development of much-needed new therapies.