Among the various polyoma virus T antigens which have so far been identified, only the large-T and a 63 000-Mr polypeptide were found to bind to double-stranded calf thymus DNA. The proteins were not retained on single-stranded DNA-cellulose columns, and a purification procedure was designed on the basis of this observation. Purified fractions (approx. 1000-fold) exhibited an enzymatic activity which converts ATP into ADP and Pi. This activity was quantitatively inhibited after preincubation in the presence of anti-(polyoma T antigen) immunoglobulins and was shown to be dependent on a virus-coded gene product (alpha gene) on the basis of the following observations: (a) ATPase activity from cells infected with tsa mutants of polyoma was reduced after a shift to the restrictive temperature; (b) the enzyme purified from tsa-infected cells maintained at the permissive temperature was more thermolabile in vitro than that prepared in parallel from cells infected with wild-type virus.