DNA polymerase was partially purified from mitochondrial extracts of rat liver by phosphocellulose, DEAE-cellulose, heparin-Sepharose CL-6B and DNA-agarose column chromatography. By these purification steps, DNA polymerase and proliferating cell nuclear antigen (PCNA) were completely separated at the step of heparin-Sepharose CL-6B column chromatography. The isolated DNA polymerase was inhibited by ddTTP, but not by aphidicolin. The enzyme sedimented at about 8 S on 5-20% analytical sucrose density gradient centrifugation. These data showed that the DNA polymerase isolated from mitochondria is gamma in type. After the separation of DNA polymerase gamma and PCNA, the two fractions were remixed and DNA polymerase gamma activity was measured. DNA polymerase gamma activity was stimulated about three-fold or more in the presence of the PCNA fraction. This stimulation was inhibited by the addition of anti-PCNA rabbit IgG2a. In addition, highly purified human recombinant PCNA stimulated the DNA polymerase gamma activity. These results indicate that DNA polymerase gamma, like DNA polymerase delta, is activated by PCNA.