The major function of the regulatory (R) subunit of the cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R.C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RII) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RII and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RII in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RII and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RII could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RII.cAMP was increased by addition of the C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RII subunit may interact with the C subunit in a cAMP-dependent manner.