The mitochondrial DNA in Crithidia fasciculata, a trypanosomatid parasite, is known as kinetoplast DNA. Kinetoplast DNA has a very unusual structure, consisting of several thousand minicircles and a few dozen maxicircles, all topologically interlocked into a giant network. There is one network within each cell's single mitochondrion. We previously purified a 43-kDa DNA polymerase from C. fasciculata mitochondria (Torri, A. F., and Englund, P. T. (1992) J. Biol. Chem. 267, 4786-4792). This enzyme has properties very different from those of a DNA polymerase gamma, the conventional mitochondrial polymerase. In addition to its small size, it is nonprocessive, has no detectable exonuclease activity, and has very low fidelity. In all of these respects, the polymerase resembles a DNA polymerase beta, a gap-filling enzyme thought to function in DNA repair in the nucleus of other eukaryotes. We speculate that this enzyme may have been specially imported into the C. fasciculata mitochondrion to repair the many gaps found in minicircles following their replication. This is the first example of a beta-like polymerase from the mitochondrion of any eukaryote.