In its physiological state, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RII beta(2):C-2 holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the beta(4)-beta(5) loop in the RII beta subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RII beta subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RII beta tetramer differs appreciably from our model of the RI alpha tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.