The two major phosvitin-utilizing kinases have been purified from virions of the Prague C strain of Rous sarcoma virus by the use of ion-exchange and affinity chromatography. The two kinases isolated may be differentiated by their molecular weights as well as by their ability to utilize GTP as a phosphate donor. Protein kinase G, which will use either GTP or ATP as a phosphate donor, has a molecular weight of 120 000 as determined under nondenaturing conditions by glycerol gradient centrifugation and 28 000 when assayed under denaturation in sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels. Protein kinase A, which will only efficiently use ATP as the phosphate donor, has an apparent molecular weight of 43 000 estimated by glycerol gradient sedimentation and 40 000 by NaDodSO4-polyacrylamide electrophoresis. Both kinases possess the ability to autophosphorylate. Phosvitin is the major, and casein the minor, phosphate-accepting substrate for both kinases in vitro; however, kinase G will also phosphorylate histones to an extent similar to that observed with casein.