A wide variety of viruses require the transient presence of scaffolding proteins to direct capsid assembly. In the case of bacteriophage P22, a model in which the scaffolding protein selectively stabilizes on-pathway growing intermediates has been proposed. The stoichiometry and thermodynamics of binding of the bacteriophage P22 scaffolding protein within the procapsid were analyzed by light scattering and isothermal titration calorimetry. Calorimetric experiments carried out between 10 and 37 °C were consistent with the presence of at least two distinct populations of binding sites, in agreement with kinetic evidence obtained by a light scattering assay. Binding to the high-affinity sites occurred at 20 °C with a stoichiometry of approximately 60 scaffolding molecules per procapsid and an apparent Kd of approximately 100-300 nM and was almost completely enthalpy-driven. For the second binding population, precise fitting of the data was impossible due to small heats of binding, but the thermodynamics of binding were clearly distinct from the high-affinity phase. The heat capacity change (ΔCp) of binding was large for the high-affinity sites and negative for both sets of sites. Addition of sodium chloride (1 M) greatly reduced the magnitude of the apparent ΔH, in agreement with previous evidence that electrostatic interactions play a major role in binding. A mutant scaffolding protein that forms covalent dimers (R74C/L177I) bound only to the high-affinity sites. These data comprise the first quantitative measurements of the energetics of the coat protein/scaffolding protein interaction.