The problem of protein stability is addressed with spectroscopic studies of equilibrium and kinetic properties of the binding of fluorescein to high-affinity monoclonal anti-fluorescyl antibodies (Mab 4-4-20), Fab fragments, and single-chain antibodies (SCA). SCA molecules contain only the variable domains of the antibody and an amino acid linker. The influence of glycerol on the antigen binding reaction is studied by circular dichroism, fluorescence, and absorption spectroscopy. The presence of glycerol in the solvent lowers the affinity of SCA for the ligand drastically, and the affinity even decreases toward lower temperatures. This effect is not observed in Fab and Mab. Analysis of the temperature jump kinetics shows that the dissociation reaction can be modeled as a two-state transition. The CD spectra indicate that the domain structure of the SCA remains unaltered in the presence of glycerol. Therefore, it is concluded that glycerol promotes the dissociation of the two variable domains of SCA. In Fab and Mab, the constant domains provide additional stabilization of the molecular structure at the antigen binding site.