The association of synthetic receptors to target guests often proceeds through the cooperative action of multiple binding forces. An investigation into the thermodynamic origin of cooperativity in ion-pairing host-guest binding in water is described. The binding affinities of 1,2,3,4-butanetetracarboxylate, tricarballate, glutarate, and acetate to a C-3v symmetric metallo-host (1) are characterized in terms of the binding constants (K-a) and the thermodynamic parameters DeltaGdegrees, DeltaHdegrees, and DeltaSdegrees, as determined by isothermal titration calorimetry (ITC). These values are used to determine the individual contributions of the binding interaction to the overall binding. Several ways to view the combination of the individual binding events that make up the whole are analyzed, all of which lead to the conclusion of negative cooperativity. Combined, the data were used to evaluate the thermodynamic origin of negative cooperativity for this series of guests, revealing that entropy is the largest contributing factor. An interpretation of this result focuses upon differences in the number of water molecules displaced upon binding.