Hammond behavior, in which two neighboring states move closer to each other along the reaction coordinate as the energy difference between them becomes smaller, has previously been observed for the transition state of unfolding of barnase. Here, we report Hammond behavior for the small protein chymotrypsin inhibitor 2 (CI2), which folds and unfolds via a single rate-determining transition state and simple two-state kinetics. Mutants have been generated along the entire sequence of the protein and the kinetics of folding and unfolding measured as a function of concentration of denaturant. The transition state was found to move progressively closer to the folded state on destabilization of the protein by mutation. Different regions of CI2 all show a similar sensitivity to changes in the energy of the transition state. This is in contrast to the behavior of barnase on mutation for which the position of the transition state for its unfolding is sensitive to mutation in some regions, especially in its major alpha-helix, but not in others. The transition state for the folding and unfolding of CI2 resembles an expanded version of the folded state and is formed in a concerted manner, in contrast to that for barnase, in which some regions of structure are fully formed and others fully unfolded. The reason for the general sensitivity of the position of the transition state of CI2 to mutation is presumably the relatively uniform degree of structure formation in the transition state and the concerted nature of its formation.(ABSTRACT TRUNCATED AT 250 WORDS)