The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 degrees C are driven by dominant favorable enthalpic contributions between 20 and 42 degrees C, with a large negative heat capacity (DeltaC(p)) of -421 +/- 27 cal mol(-1) K(-1). The unfavorable negative value of entropy (DeltaS(b)(0)) at 25 degrees C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 degrees C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (DeltaDeltaG(b)(0) congruent with 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC:B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site.