We have purified the 36 and 63 kd cellular proteins known to associate with polyomavirus middle and small tumor (T) antigens and SV40 small t antigen. Microsequenclng of the 36 kd protein indicated that it was probably identical to the catalytic subunit of protein phosphatase 2A (PPSA). Identity was confirmed by comigration on two-dimensional(2D) gels and by 20 analysis of complete chymotryptic digests. In addition, PPSA-like phosphatase activity was detected in immunoprecipitates of wild-type middle T. Immunoblotting experiments, comigration on 2D gels, and 20 analysis of limit chymotryptic digests demonstrated that the 63 kd protein, present in the middle T complex in approximately equimolar ratio to the 36 kd protein, is a known regulatory subunit of the PPSA holoenzyme. Finally, the 36 kd PPSA catalytic subunit can be immunoprecipitated by anti-pp60c-src antisera only from cells expressing wild-type middle T. These results suggest that complex formation between PPSA and T antigens may be important for T antigen-mediated transformation.