Rat liver mitoplasts (inner mitochondrial membrane and matrix) contain protein kinase activity. This activity increases twofold on addition of Triton X-100. The activity observed in absence of Triton X-100 is probably exposed on the outer surface of mitoplasts, since it is sensitive to trypsin treatment. Most of the remaining protein kinase is bound to the membrane fraction, presumably on the inside of (or else hidden in) the inner mitochondrial membrane. Only a small part of the kinase activity is found in the mitochondrial matrix. A phosphoprotein band, partly resolved into a doublet, was observed on electrophoresis in SDS-polyacrylamide gels after endogeneous phosphorylation of mitoplasts, inner mitochondrial membrane or matrix. When isolated fractions are phosphorylated approximately 75% of the phosphoprotein is found in the matrix, and the remainder in the inner membrane. The phosphorylation of the doublet is inhibited by inhibitors to pyruvate dehydrogenase kinase, suggesting that it represents the phosphorylated subunit of pyruvate dehydrogenase.