Endogenous rat liver substrate proteins for adenosine 3′, 5′-monophosphate (cyclic AMP*)-dependent protein kinase [EC 2.7.1.37] were analyzed by means of poly-acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The substrate proteins were assayed in terms of their ability to accept the terminal phosphate of ATP in the presence of protein kinase obtained from the soluble fraction of the same tissue. A number of substrate proteins were found in all subcellular fractions, including cytosol, microsomes, mitochondria, and nuclei. Plasma membranes also contained phosphate acceptors. Among nuclear proteins, not only histone but also nonhistone proteins were active as substrates. The existence of numerous substrate proteins within the cell is compatible with the view that cyclic AMP-dependent protein kinase may play a role in various processes controlled by cyclic AMP in this tissue.