Two protein kinases (protein kinases A1 and A2) [EC 2. 7.1.37] were partially purified from rat liver chromatin by salt extraction followed by Sephadex G-200 gel nitration and phosphocellulose column chromatography. These kinases catalyze the transfer of the terminal phosphate of ATP to specific seryl and threonyl residues of nuclear non-histone phosphoproteins. Using sodium dodecyl sulfate-polyacrylamide gel electro-phoresis the phosphoproteins were shown to be heterogeneous; each protein kinase phosphorylates different protein species. Casein and phosvitin also serve as effective substrates, but histone and protamine are less than 7% as active as casein. Adenosine 3′;, 5′-monophosphate does not stimulate or inhibit these enzymes. The molecular weight was estimated by the gel filtration procedure to be 2.3 x 105 for protein kinase A1 and 4.0x104 for protein kinase A2. Protein kinase A1 and A2 show Kmvalues for ATP of 6x10−6M and lxlO−5M, Kmvalues for casein of 1.2g/liter and 0.4g/ liter; and pH optima of about 7.5 and 8.0, respectively. By subcellular fractionation with non-aqueous solvents, these protein kinases were shown to be localized also in cytoplasm although the specific activities are one-twentieth of that found in chromatin.