The crystallographic structure of a complex between cyclosporin A and the Fab fragment of monoclonal antibody R45-45-11 has been solved to 2.65 A resolution (Altschuh et al., 1992a, Science 256, 92; Vix et al., 1993, Proteins 15, 339), yielding a precise three-dimensional picture of interacting surfaces. In order to evaluate the contribution of observed contacts to the energy of interaction, we have measured the effect on binding affinity of minor chemical modifications of CS. The equilibrium binding constant of the Fab fragment for a set of cyclosporin analogs was obtained by measuring in a biosensor instrument the dependence of complex formation on Fab concentration, at constant analog concentrations. Data were analysed using Scatchard plots. Differences in binding energy resulting from cyclosporin modifications discriminated between two types of contact areas. The first type displays adaptability to structural modifications of cyclosporin at the cost of a small decrease in binding energy, and contacting residues in the antibody form the periphery of the combining site. The second type does not accommodate structural changes and corresponds in cyclosporin to three residues whose modifications drastically decrease binding energy with the antibody. The corresponding contact residues in the antibody form the core of the antibody combining site.