To better understand the molecular mechanism of cAMP-induced and substrate-enhanced activation of type-I A-kinase, we measured the kinetics of A-kinase regulatory subunit interactions using a stopped-flow spectrofluorometric method. Specifically, we conjugated fluorescein maleimide (FM) to two separate single cysteine-substituted and truncated mutants of the type Ialpha regulatory subunit of A-kinase, RIalpha (91-244). One site of cysteine substitution and conjugation was at R92 and the other at R239. Although the emission from both conjugates changed with catalytic subunit binding, only the FM-R92C conjugate yielded unambiguous results in the presence of cAMP and was therefore used to assess whether a pseudosubstrate perturbed the rate of holoenzyme dissociation. We found that cAMP selectively accelerates the rate of dissociation of the RIalpha (91-244):C-subunit complex approximately 700-fold, resulting in an equilibrium dissociation constant of 130 nM. Furthermore, excess amounts of the pseudosubstrate inhibitor, PKI(5-24), had no effect on the rate of RIalpha (91-244):C-subunit complex dissociation. The results indicate that the limited ability of cAMP to induce holoenzyme dissociation reflects a greatly reduced but still significant regulatory catalytic subunit affinity in the presence of cAMP. Moreover, the ability of the substrate to facilitate cAMP-induced dissociation results from the mass action effect of excess substrate and not from direct substrate binding to holoenzyme.