Publisher Summary This chapter discusses the radioimmunoassay of the regulatory subunit of type I adenosine 3',5'-monophosphate (cAMP) dependent protein kinase. Much evidence has accumulated, suggesting that the mechanisms of action of cAMP in mammalian tissues are mediated by cAMP-dependent protein kinases. There are two different forms of cAMP-dependent protein kinases that have been identified in the mammalian tissues. These are termed as type I and type II cAMP-dependent protein kinases based on their elution profiles on diethylethanolamine (DEAE)-cellulose chromatography. The R-cAMP complex of bovine skeletal muscle and bovine heart muscle are prepared by utilizing DEAE-cellulose fractionation and immobilized N 6 H 2 N(CH 2 ) 2 -cAMP affinity chromatography. The phosphotransferase activity of cAMP-dependent protein kinase is determined in the presence of 1.2 μM cAMP. It is found that the cyclic nucleotide-free beef skeletal muscle RI u , plotted as a semilogarithmic function from 0.003 to 10 pmol concentrations, competed in a concentration-dependent manner for the RI antiserum binding to [ 125 I]RI u . The competitive cross-reactivity of various rat tissue protein kinase extracts in the RI radioimmunoassay is also described in this chapter.