Protein kinase I from rabbit skeletal muscle binds adenosine 3':5'-monophosphate (cAMP) and ATP with high affinity; cAMP promotes and ATP retards dissociation (activation) of the tetrameric enzyme. The interrelationship of ligand interaction with protein dissociation has been probed by quantitative ligand binding, using the filter assay technique, and by computer simulation of binding curves in terms of Scatchard plots. A comparison of the experimental and computed binding data strongly confirms the supposition that the dimeric regulatory subunit (R2) binds cAMP cooperatively. Mainly from ATP binding it is further concluded that the interaction of the two catalytic subunits with R2 is also strongly cooperative, whereas the binding of ATP to the holoenzyme is non-cooperative. The underlying random model, possessing low-affinity and high-affinity sites not only for ATP but also for cAMP, allows the determination of a minimal set of equilibrium parameters required for description and also an estimation of their magnitude. It further provides a basis for an explanation of the different behaviour of protein kinases I and II reported in the literature.