Complex formation of large-ring cyclodextrins (CDs) having DP21-32 with iodine in aqueous KI solution was studied by isothermal titration calorimetry (ITC). When the individual CD is shown, the term CDn is used, where n represents the degree of polymerization. For CDs in the DP range of 21-32, the curves obtained for the titration of the CDs with iodine can be analyzed by a model assuming 1:2 complex formation with identical interacting sites. For the case of two identical interacting sites, the binding constants K1 and K2 (K1＜K2), defined relative to the progress of saturation, lie in the range 0.7 to 7.3×10³ M^-1 and 3.0 to 62.6×10³ M^-1, respectively. The values of ΔH₂ and TΔS₂ lie in the range -34.9 to -136.4 kJ mol^-1 and -15.5 to -112.8 kJ mol^-1, respectively. The largest values for -TΔS₂ obtained for a CD26 can, in part, be attributed to a large decrease in conformational flexibility of the CD which occurs during complex formation. Based on ITC data, conformational changes in CD26 which are induced as the results of the formation of a CD-iodine inclusion complex are discussed in terms of the recently reported crystal structure of CD26.