cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R- and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar Ka's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.