(RP)-cAMPS is known to inhibit competitively the cAMP-induced activation of cAMP-dependent protein kinase (PKA). The molecular nature of this inhibition, however, is unknown. By monitoring the intrinsic tryptophan fluorescence of recombinant type I regulatory subunit of PKA under unfolding conditions, a free energy value (delta GDH2O) of 8.23 +/- 0.22 kcal/mol was calculated. The cAMP-free form of the regulatory subunit was less stable with delta GDH2O = 6.04 +/- 0.05 kcal/mol. Native stability was recovered by treatment of the cAMP-free protein with either cAMP or (SP)-cAMPS but not with (RP)-cAMPS. Thus, (RP)-cAMPS binding to the regulatory subunit keeps the protein in a locked conformation, unable to release the catalytic subunit. This finding was further supported by demonstrating that holoenzyme formation was greatly accelerated only when bound cAMP was replaced with (RP)-cAMPS but not with cAMP or (SP)-cAMPS.