The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The α and β2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the αβ isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the α and β2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the αβ polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the α, β2, and αβ molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.