The interaction of barnase, an extracellular RNase of Bacillus amylolique-faciens, with its intracellular inhibitor barstar is a suitable paradigm for protein-protein interactions, since the structures of both the free and the complexed proteins are available at high resolution. The contributions of residues from both proteins to the energetics of kinetics and thermodynamics of binding were measured by double mutant cycle analysis. Such cycles reveal whether the contributions from a pair of residues are additive, or the effects of mutations are coupled. The aim of the study was to determine which of the interactions are co-operative. Double mutant cycles were constructed between a subset of five barnase and seven barstar residues, which were shown by structural and mutagenesis studies to be important in stabilising the complex. The coupling energy between two residues was found to decrease with the distance between them. Generally, residues separated by less than 7 A interact co-operatively. At greater separations, the effects of mutation are additive, and the energetics of the interactions are independent of each other. The highest coupling energies are found between pairs of charged residues (1.6 to 7 kcal mol-1). Three of the six most important interactions detected by double mutant cycle analysis (with coupling energies of more than 3.0 kcal mol-1) had not been noted previously from examination of the crystal structure. The effects of mutation on the kinetics of association are all additive, apart from charged residues located at distances of up to 10 A apart, which are co-operative. This can be explained by the fact that the transition state for association occurs before most interactions are formed.