In higher eukaryotes, DNA polymerase (pol) beta resides in the nucleus and participates primarily in DNA repair. The DNA polymerase beta from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol beta-like proteins. One is approximately 70% identical to the C. fasciculata pol beta and is likely the homolog of this enzyme. The other, although approximately 30% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different.