The phosphorylation pattern in mitochondrial fractions isolated from hepatocytes, preincubated with 32P-phosphate and stimulated with glucagon and calcium mobilizing hormones, was studied. Only in mitochondria from glucagon treated hepatocytes two phosphorylated protein bands were observed, one with a molecular weight (MW) of 54 kDa in the outer membrane fraction which, according to the literature, is suggested to represent protein kinase A; one with a MW of 20 kDa in the inner membrane fraction which has not been described earlier. Electroelution and digestion of the 20 kDa protein band yielded two tryptic peptides which were identified as fragments homologous to human cytokeratin type II (the sequence of rat cytokeratin type II is not known). From the amino acid composition and sequence, and from the known structure of type II cytokeratins, it is concluded that the 20 kDa phosphoprotein is composed of amino- and carboxylterminal proteolytic fragments of rat cytokeratin C8 which are tightly anchored in the inner mitochondrial membrane. The physiological significance of the possible interaction of cytoskeletal proteins with the mitochondrial inner membrane and its hormonal regulation are discussed.