Trypanosoma brucei and related parasites are causative agents of severe diseases that affect global health and economy. T. brucei is responsible for sleeping sickness in humans (African trypanosomiasis) and a wasting disease in livestock. More than 100 years after T. brucei was identified as the etiological agent for sleeping sickness, available treatments remain inadequate, complicated by toxicity, lengthy and expensive administration regiments, and drug-resistance. There is clear need for the development of a new antitrypanosomal drugs. Due to the unique evolutionary position of these early diverging eukaryotes, trypanosomes posses a number of biological properties unparalleled in other organisms, including humans, which could prove valuable for new drug targets. One of the most distinctive properties of trypanosomes is their mitochondrial DNA, called kinetoplast DNA (kDNA). kDNA is composed of over five thousand circular DNA molecules (minicircles and maxicircles) catenated into a topologically complex network. Replication of kDNA requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication and at least five DNA polymerases. Three of these (POLIB, POLIC, and POLID) are related to bacterial DNA polymerase I and are required for kDNA maintenance and growth. Each polymerase appears to make a specialized contribution to kDNA replication. The research described in this dissertation is a significant contribution to the field of kDNA replication and the advancement of kDNA replication proteins as putative drug targets for sleeping sickness. Functional characterization of POLIB indicated that it participates in minicircle replication but is likely not the only polymerase contributing to this process. Gene silencing of POLIB partially blocked minicircle replication and led to the production of a previously unidentified free minicircle species, fraction U. Characterization of fraction U confirmed its identity as a population of dimeric minicircles with non-uniform linking numbers. Fraction U was not produced in response to silencing numerous other previously studied kDNA replication proteins but, as we demonstrated here, is also produced in response to POLID silencing. This common phenotype led us to hypothesize that POLIB and POLID both participate in minicircle replication. Simultaneously silencing both polymerases completely blocked minicircle replication, supporting a model of minicircle replication requiring both POLIB and POLID. Finally, we demonstrate that disease-causing trypanosomes require kDNA and the kDNA replication proteins POLIB, POLIC, and POLID. These data provide novel insights into the fascinating mechanism of kDNA replication and support the pursuit of kDNA replication proteins as novel drug targets for combating African trypanosomiasis.