We used electron microscopy to examine the structure of human DNA pol gamma, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of negatively stained preparations of the catalytic subunit, pol gammaA, and of the holoenzyme including a dimeric accessory factor, pol gammaB(2), permitted unambiguous identification of the position of the accessory factor within the holoenzyme. The model explains protection of a partial chymotryptic cleavage site after residue L(549) of pol gammaA upon binding of the accessory subunit. This interaction region is near residue 467 of pol gammaA, where a disease-related mutation has been reported to impair binding of the B subunit. One pol gammaB subunit dominates contacts with the catalytic subunit, while the second B subunit is largely exposed to solvent. A model for pol gamma is discussed that considers the effects of known mutations in the accessory subunit and the interaction of the enzyme with DNA.