pp60src, the product of the rous sarcoma virus src gene, was purified greater than 100,000-fold by a combination of ion-exchange and immunoaffinity chromatography. Incubation of pp60src purified in this fashion with [32P-gamma]ATP resulted in a single 32P-labeled protein when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staining of these gels with silver nitrate showed a predominant 60,000-dalton polypeptide which comigrated with the protein labeled with 32P in vitro. Partial digestion of this protein with V8 protease after in vitro iodination indicated that it was pp60src. These results suggest that pp60src is able to autophosphorylate.