DNA polymerase gamma and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase gamma and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements of Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) - (dT)20-25 larger than activated DNA larger than poly(dA) - (dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA; molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pI 5.4). We therefore conclude that brain nuclear DNA polymerase gamma and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.