A study of the phosphorylated sites on the regulatory subunit of type II cyclic AMP-dependent protein kinase is described. The site of autophosphorylation, previously reported (Takio, K., Walsh, K. A., Neurath, H., Smith, S. B., Krebs, E. G., and Titani, K. (1980) FEBS Lett. 114, 83-88), is found to contain the only residue phosphorylated by the catalytic subunit. Endogenous protein-bound phosphate, present at a second site, is observed in the NH2-terminal region of purified type II regulatory subunit. A serine residue in this same domain of type II regulatory subunit is shown to be phosphorylated in vitro by casein kinase II. The stoichiometry of phosphate incorporation by casein kinase II indicates a single site of phosphorylation that is equivalent to the site containing the endogenous phosphate. The apparent Km and Vmax of casein kinase II for the phosphorylation of purified type II regulatory subunit containing 0.55 molar equivalents of endogenous phosphate are 36 microM and 3.5 mumol/min/mg, respectively. Phosphatase treatment of type II regulatory subunit decreased the determined Km to 13 microM but does not change the Vmax consistent with the suggestion that casein kinase II is phosphorylating a site partially occupied by endogenous phosphate. Comparison of peptide maps of type II regulatory subunit phosphorylated in vitro by casein kinase II with in vivo modified type II regulatory subunit indicates that the site modified in vitro is also phosphorylated in vivo.