Common causes of human mitochondrial diseases are mutations affecting DNA polymerase (Pol) gamma, the sole polymerase responsible for DNA synthesis in mitochondria. Although the polymerase and exonuclease active sites are located on the catalytic subunit Pol gammaA, in holoenzyme both activities are regulated by the accessory subunit Pol gammaB. Several patients with severe neurological and muscular disorders were reported to carry the Pol gammaA substitutions R232G or R232H, which lie outside of either active site. We report that Arg(232) substitutions have no effect on independent Pol gammaA activities but show major defects in the Pol gammaA-Pol gammaB holoenzyme, including decreased polymerase and increased exonuclease activities, the latter with decreased selectivity for mismatches. We show that Pol gammaB facilitates distinguishing mismatched from base-paired primer termini and that Pol gammaA Arg(232) is essential for mediating this regulatory function of the accessory subunit. This study provides a molecular basis for the disease symptoms exhibited by patients carrying those substitutions.