Site-directed mutagenesis has been employed to substitute cysteine for valine at position 55, which is located on the dimer interface of the Cro protein of bacteriophage lambda. It has been found that the Cys55 Cro protein (Cro VC55) spontaneously forms a stable disulfide-bonded dimer in the absence of a reducing agent. UV-CD and NMR data showed that the mutant protein retains the conformation of the wild Cro protein and has acquired significant heat-stability. However, its specific DNA-binding activity is reduced several times compared with that of the wild Cro. Photochemically induced dynamic nuclear polarization (CIDNP) spectra demonstrated that a conformational change of Cro VC55 did not take place upon the formation of a complex with OR3, in contrast to the case of the wild Cro. These data suggest that the induced fitting, like loosening, of the two subunits of the wild Cro dimer contributes to the enhancement of its affinity to its operator DNA, which results in a specific interaction between Cro and OR3.