Complex formation of large cyclodextrins(CDs) having DP 21-32 with iodine in aqueous KI solution was studied by isothermal titration calorimetry. The curves obtained for the titration of the CDs with iodine cannot be analyzed by a model based on a single set of identical sites, but, rather, by a model assuming 1:2 complex formation with identical interacting sites. For the two identical interacting sites, the binding constants K-1 and K-2 (K-1 < K-2), defined relative to the progress of saturation, lie in the range 0.7 to 7.3 x 10(3) M-1 and 3.0 to 62.6 x 10(3) M-1, respectively. The values of Delta H-2 and T Delta S-2 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 of -T Delta S-2 obtained for a CD of DP 26 can, in part, be attributed to a large decrease in conformational flexibility of the CD which occurs during complex formation.