The core protein (HBcAg) of hepatitis B virus (HBV) has been shown to interact with the large surface antigen during HBV morphogenesis, and these interactions can be blocked by small peptides selected from either linear or constrained phage display peptide libraries. The association of HBcAg with peptide inhibitors was quantitatively evaluated by isothermal titration calorimetry. The thermodynamic data show that the interaction between HBcAg and peptide MHRSLLGRMKGA is enthalpy-driven and occurs at a 3:1 stoichiometry and dissociation constant (Kd) value of 79.4 muM. However, peptide WSFFSNI displays a higher binding affinity for HBcAg with a Kd value of 18.5 muM when compared to peptide MHRSLLGRMKGA. A combinatorial approach using chemical cross-linking and surface-enhanced laser desorption/ionization-time-of-flight-mass spectrometry shows that the Lys of peptide MHRSLLGRMKGA interacted either with D64, E77, or D78 of HBcAg.