Rodent monoclonal antibodies have been "humanized" or "reshaped" for therapy by transplanting the antigen-binding loops from their variable domains onto the beta-sheet framework regions of human antibodies. However, additional substitutions in the human framework regions are sometimes required for high affinity antigen binding. Here we describe antigen binding by a reshaped antibody derived from the mouse anti-lysozyme antibody D1.3, and several variants in which point mutations had been introduced into framework positions to improve its affinity. The affinities were determined from the relaxation kinetics of reactant mixtures using quenching of fluorescence that occurs upon formation of the antibody-antigen complex. The dissociation constant of lysozyme ranged from 3.7 nM (for D1.3) to 260 nM. Measurement of antibody-antigen association kinetics using stopped-flow showed that D1.3 and most of the reshaped antibodies had bimolecular rate constants of 1.4 x 10(6) s-1 M-1, indicating that differences in equilibrium constant were predominantly due to different rates of dissociation of lysozyme from immune complexes. Mutations in a triad of heavy chain residues, 27, 29 and 71, contributed 0.9 kcal/mol in antigen binding free energy, and a Phe to Tyr substitution of light chain residue 71 contributed an additional 0.8 kcal/mol. The combined effect of all these mutations brought the affinity of the reshaped antibody to within a factor of 4 of D1.3. All of these substitutions were in the beta-sheet framework closely underlying the complementarity-determining regions, and do not participate in a direct interaction with antigen. The informed selection of residues in such positions may prove essential for the success of loop transplants in antibodies. Variation of these sites may also have a role in shaping the diversity of structures found in the primary repertoire, and in affinity maturation.