Background: Mutations in the human mitochondrial DNA polymerase (Pol-) have been linked to diseases with varying severity and age of onset. Results: Yeast cells expressing human Pol- reveal a correlation of Pol- fidelity with human disease onset. Conclusion: Humanized yeast provides an efficient system to correlate biochemical defects in Pol- with physiological consequences. Significance: The Pol--associated diseases may be caused by the low accuracy of Pol- mutants, not low rates of replication. Mutations in the human mitochondrial polymerase (polymerase- (Pol-)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol- with their physiological consequences, we created humanized yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol- suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol- mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans.