Protein kinase A (PKA) isozymes are distinguishable by the inhibitory pattern of their regulatory (R) subunits with RI subunits containing a pseudophosphorylation P(0)-site and RII subunits being a substrate. Under physiological conditions, RII does not inhibit PrKX, the human X chromosome encoded PKA catalytic (C) subunit. Using a live cell Bioluminescence Resonance Energy Transfer (BRET) assay, Surface Plasmon Resonance (SPR) and kinase activity assays, we identified the P(0)-position of the R subunits as the determinant of PrKX autoinhibition. Holoenzyme formation only takes place with an alanine at position P(0), whereas RI subunits containing serine, phosphoserine or aspartate do not bind PrKX. Surprisingly, PrKX reversibly associates with RII when changing P(0) from serine to alanine. In contrast, PKA-Calpha forms holoenzyme complexes with all wildtype and mutant R subunits; however, holoenzyme re-activation by cAMP is severely affected. Only PKA type II or mutant PKA type I holoenzymes (P(0): Ser or Asp) are able to dissociate fully upon maximally elevated intracellular cAMP. The data are of particular significance for understanding PKA isoform-specific activation patterns in living cells.