Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-10/HEWL complex demonstrates that the energetically important side chains (hot spots) of both partners are in contact. A plot of ΔΔGHyHEL-10_mutant vs. ΔΔGHEWL_mutant for the five of six interacting side-chain hydrogen bonds is linear (Slope = 1). Only 3 of the 13 residues in the HEWL epitope contribute >4 kcal/mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32H/K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32HN/K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the ΔΔG values of the Tyr → Phe vs. the corresponding Tyr → Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total ΔΔG observed for the Tyr → Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31L/K96 hydrogen bond with a salt bridge, N31DL/K96, destabilizes the complex by 1.4 kcal/mol. The free energy of interaction, ΔΔGint, obtained from double mutant cycle analysis showed that ΔΔGint for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large ΔΔG values are analyzed.